Methods of assessing the need for and the effectiveness of therapy with antioxidants

ABSTRACT

The invention relates to diagnostic methods for assessing the need of a subject for treatment with an anti-oxidant, or alternatively, for determining the utilization efficiency and ultimate effectiveness of anti-oxidant therapy in subjects having been treated with antioxidants. More specifically, the methods of the present invention are particularly useful in prophylactic assessment of individuals at risk for developing diseases or conditions in which oxidative stress plays a role, such that an appropriate therapeutic regimen can be prescribed for that individual, thus leading to alternative therapies and/or life style changes. The invention further relates to methods for assessing the need for, the utilization efficiency and the effectiveness of therapy in subjects having received therapy with specific antioxidant and immune enhancing formulations. Kits are also provided for measuring the levels of markers of oxidative stress and immune cell numbers.

FIELD OF THE INVENTION

The invention relates to diagnostic methods for assessing the need of asubject for treatment with an anti-oxidant, or alternatively, fordetermining the effectiveness of anti-oxidant therapy in subjects havingbeen treated with antioxidants. More specifically, it relates to methodsfor assessing the need for, or effectiveness of therapy in subjectshaving received therapy with, specific antioxidant and immune enhancingformulations.

BACKGROUND OF THE INVENTION

It is generally recognized that many disease processes are attributed tothe presence of elevated levels of free radicals and reactive oxygenspecies (ROS) and reactive nitrogen species (RNS), such as superoxide,hydrogen peroxide, singlet oxygen, peroxynitrite, hydroxyl radicals,hypochlorous acid (and other hypohalous acids) and nitric oxide.

In the eye, cataract, macular degeneration and degenerative retinaldamage are attributed to ROS. Among other organs and their ROS-relateddiseases include: lung cancer induced by tobacco combustion products andasbestos; accelerated aging and its manifestations, including skindamage and scleroderma; atherosclerosis; ischemia and reperfusioninjury, diseases of the nervous system such as Parkinson disease,Alzheimer disease, muscular dystrophy, multiple sclerosis; lung diseasesincluding emphysema and bronchopulmonary dysphasia; iron overloaddiseases such as hemochromatosis and thalassemia; pancreatitis;diabetes; renal diseases including autoimmune nephrotic syndrome andheavy metal-induced nephrotoxicity; and radiation injuries. Diseases ofaging and chronic emotional stress also appear to be associated with adrop in glutathione levels, which allows ROS to remain active.

Certain anti-neoplastic drugs such as adriamycin and bleomycin inducesevere oxidative damage, especially to the heart, limiting the patient'sexposure to the drug. Redox-active metals such as iron induce oxidativedamage to tissues; industrial chemicals and ethanol, by exposure andconsumption, induce an array of oxidative damage-related injuries, suchas cardiomyopathy and liver damage. Airborne industrial andpetrochemical-based pollutants, such as ozone, nitric oxide, radioactiveparticulates, and halogenated hydrocarbons, induce oxidative damage tothe lungs, gastrointestinal tract, and other organs. Radiation poisoningfrom industrial sources, including leaks from nuclear reactors andexposure to nuclear weapons, are other sources of radiation and radicaldamage. Other routes of exposure may occur from living or working inproximity to sources of electromagnetic radiation, such as electricpower plants and high-voltage power lines, x-ray machines, particleaccelerators, radar antennas, radio antennas, and the like, as well asusing electronic products and gadgets which emit electromagneticradiation such as cellular telephones, and television and computermonitors.

Mammalian cells have numerous mechanisms to eliminate these damagingfree radicals and reactive species. One such mechanism includes theglutathione system, which plays a major role in direct destruction ofreactive oxygen compounds.

Perhaps one of the most important contributions of glutathione tomammalian health is its participation in the proper functioning of theimmune system to respond to infection or other types of trauma. It isknown that weakening of the immune system caused by infection or othertraumas occurs concurrently with depletion of glutathione in bodytissues. It is known, also, that such weakening can be reversed byreplenishing the body's level of glutathione by intracellular synthesis.It is believed that glutathione accomplishes its salutary effects byprotecting immune cells against the ravages of oxidizing agents and,free radicals.

Until recently, the lack of specific and dependable methods forevaluating oxidant stress in vivo made it very difficult to establish acause and effect relationship between free radical-generating agents orconditions and disease pathology. Furthermore, the various treatmentstrategies with anti-oxidants have been difficult to monitor due to thelack of techniques sufficiently sensitive to reliably provide an indexof oxidative damage in vivo.

For example, there is currently substantial evidence that oxidation ofLDL occurs in vivo, and results of animal studies suggest that this maylead to the formation and build up of atherosclerotic plaques. Althoughepidemiological data support a role for antioxidants in the preventionof clinical events, intervention trials thus far have given mixedresults (Steinberg D, Witztum J L Lipoproteins, lipoprotein oxidation,and atherogenesis. In Chien K R, ed. Molecular Basis of CardiovascularDisease. Philadelphia, Pa: W.B. Saunders Co., 1998:458-475). This may bedue, in part, to the fact that until now techniques to adequatelyprovide an index of in vivo lipid peroxidation have not been available,which could be-used to design and monitor effective antioxidantintervention trials to adequately test the oxidation hypothesis.

Furthermore, there are no set measures to identify high-risk groups thatwould theoretically benefit most from antioxidant therapies orinterventions. Additionally, there are no reliable means to measure ordetermine the effectiveness of such interventions in vivo. In theabsence of such methodology, current (and future) clinical trialstesting natural (or synthetic) antioxidants, which utilize clinicalendpoints, may give incorrect conclusions regarding the role ofantioxidants in specific disease states. This is a possibility becauseof the inclusion of populations that would not be expected to benefitfrom antioxidant supplementation, and/or because the dose or agentyielded insufficient antioxidant protection.

It is with respect to the development of more sensitive and accurateassays for assessing the need for intervention with anti-oxidant therapyand for monitoring the effectiveness and utilization efficiency of novelanti-oxidants that the current invention is directed.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention relates to methods forassessing the need of a subject for treatment with an antioxidant, oralternatively, if a subject is currently being treated with ananti-oxidant, the invention provides for measuring the utilizationefficiency of the anti-oxidant and the subsequent effectiveness oftherapy with the anti-oxidant. It is also an object of the presentinvention to provide a means for determining the anti-oxidativeeffective amounts of specific anti-oxidant formulations for delivery toa subject in need of such therapy. More particularly, the inventionprovides for methods for determining the amount of anti-oxidantsnecessary to increase glutathione synthesis or re-synthesis in a patientin need of such therapy.

Accordingly, a first aspect of the invention provides for a method forassessing the need for treatment with an anti-oxidant comprising thesteps of:

-   -   a) collecting a sample of body fluid from a subject suspected of        needing such treatment;    -   b) measuring the amount of lipid peroxide and pyroglutamic acid        (PGA) levels in said sample;    -   c) measuring the level of blood plasma glutathione;    -   d) comparing the amount of lipid peroxide and pyroglutamic acid        in said sample with that of a normal standard; and    -   e) comparing the level of blood plasma glutathione with that of        a normal standard; and        wherein the presence of lipid peroxide and pyroglutamic acid in        said sample and the blood plasma levels of glutathione are        present in amounts that lie outside the normal range are        indicative of a need for anti-oxidant treatment.

In a particular embodiment, the patient or subject is preferably ananimal, including but not limited to animals such as monkeys, cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human. In one embodiment, a non-human mammal is thesubject. In another embodiment, a human mammal is the subject. In yetanother particular embodiment, the subject in need of treatment with ananti-oxidant also experiences a reduction in immune cell number and/orfunction. In another particular embodiment, the immune cell is selectedfrom the group consisting of a T cell, a B cell or a natural killercell. In yet another particular embodiment, the T cell is selected fromthe group consisting of a CD4+ or a CD8+ T cell.

In another particular embodiment, the sample of body fluid is urine. Inanother particular embodiment, the sample of body fluid is whole blood.In a yet further particular embodiment, the sample of body fluid isplasma or serum.

In another particular embodiment, the anti-oxidant is selected from thegroup consisting of glutathione precursors, IMMUNE FORMULATION 100™ andIMMUNE FORMULATION 200™.

A second aspect of the invention provides a method for measuring theeffectiveness of therapy with an anti-oxidant in a subject comprisingthe steps of:

-   -   a) collecting a sample of body fluid from a subject being        treated with an anti-oxidant;    -   b) measuring the amount of lipid peroxide and pyroglutamic acid        in said sample;    -   c) measuring the level of blood plasma glutathione;    -   d) comparing the amount of lipid peroxide and pyroglutamic acid        in said sample with that of a normal standard;    -   e) comparing the level of blood plasma glutathione with that of        a normal standard; and        wherein the presence of normal levels of lipid peroxide and        pyroglutamic acid in said sample and the presence of normal        levels of blood plasma glutathione are an indication of        effectiveness of the anti-oxidant therapy.

In a particular embodiment, the method may further comprise determiningwhether immune cell number and/or function is normalized in the subject,wherein the normalization is indicative of the effectiveness of therapywith the anti-oxidant. In another particular embodiment, the immune cellis selected from the group consisting of a T cell, a B cell or a naturalkiller cell. The method of claim 9, wherein said T cell is selected fromthe group consisting of a CD4+ T cell or a CD8+ T cell. In anotherparticular embodiment. The method of claim 7, wherein said anti-oxidantis selected from the group consisting of glutathione precursors, IMMUNEFORMULATION 100™ or IMMUNE FORMULATION 200™. In yet another particularembodiment, the sample of body fluid is urine. In another particularembodiment, the sample of body fluid is whole blood. In a yet furtherparticular embodiment, the sample of body fluid is plasma or serum.

In another particular embodiment, the anti-oxidant is selected from thegroup consisting of glutathione precursors, IMMUNE FORMULATION 100™ orIMMUNE FORMULATION 200™.

In yet another preferred embodiment, the method further comprisesmeasurement of a secondary endpoint for monitoring the effectiveness oftherapy. For example, wherein the patient under treatment with theanti-oxidant therapy is suffering from a disease which is, in part,caused by high levels of oxidant stress, or where a particular drugtreatment itself results in oxidative damage to a particular tissue ororgan, such as with chemotherapy, it is beneficial to measure theeffectiveness of therapy with the anti-oxidant using the steps describedabove. However, in diseases such as atherosclerosis or cardiovasculardisease, whereby oxidized low density lipoprotein (LDL) has beenimplicated in the initiation and/or exacerbation of the disease process,it would be beneficial to monitor the effects of the antioxidant therapynot only on lipid peroxide, PGA and glutathione levels, but also on forexample, cardiac function to determine whether the antioxidant therapyhas effects on the sequelae of high oxidative stress levels, such ascardiovascular disease or atherosclerosis. The secondary endpoint mayinclude lowering of triglycerides, LDLs or increasing of high densitylipoproteins (HDLs), or measurement of cardiac function using standardtesting known to one skilled in the art.

A third aspect of the invention provides a method for measuring theutilization efficiency of an anti-oxidant comprising the steps of:

-   -   a) collecting samples of body fluid from a subject being treated        with an anti-oxidant each day after initiation of therapy and up        to 14 days after therapy has been initiated;    -   b) measuring the amount of lipid peroxide and pyroglutamic acid        in said samples;    -   c) measuring the level of blood plasma glutathione;    -   d) comparing the amount of lipid peroxide and pyroglutamic acid        in said samples with that of a normal standard;    -   e) comparing the level of blood plasma glutathione with that of        a normal standard; and        wherein the presence of normal levels of lipid peroxide and        pyroglutamic acid in said samples and the presence of normal        levels of blood plasma glutathione are an indication of        efficiency of utilization of the anti-oxidant.

In a particular embodiment, the method further comprises determiningwhether immune cell number and/or function is normalized in the subject,wherein the normalization is indicative of the utilization efficiency ofthe anti-oxidant. In another particular embodiment, the immune cell isselected from the group consisting of a T cell, a B cell or a naturalkiller cell. In another particular embodiment the T cell is selectedfrom the group consisting of a CD4+ or a CD8+ T cell. In yet anotherparticular embodiment, the sample of body fluid is urine. In anotherparticular embodiment, the sample of body fluid is whole blood. In a yetfurther particular embodiment, the sample of body fluid is plasma orserum.

In another particular embodiment the anti-oxidant is selected from thegroup consisting of glutathione precursors, IMMUNE FORMULATION 100™ orIMMUNE FORMULATION 200™.

In another preferred embodiment, if the levels of lipid peroxides,pyroglutamic acid, and glutathione are not normalized, the levels ofantioxidants are increased in dosage until such normalization occurs.

A fourth aspect of the invention provides a method for determining theamount of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ that isnecessary to increase glutathione synthesis or re-synthesis in a subjectin need of such therapy, comprising the steps of:

-   -   a) collecting a series of body fluid samples from a subject        suspected of being in need of such treatment, wherein said body        fluid samples are collected prior to the start of treatment, and        daily after the start of treatment for about 14 days;    -   b) measuring the amount of lipid peroxide and pyroglutamic acid        in said body fluid samples;    -   c) comparing the amount of lipid peroxide and pyroglutamic acid        in said body fluid samples with that of normal standards;    -   d) measuring the amount of glutathione increase in blood        samples;    -   e) comparing the amount of glutathione in said blood samples        with that of normal standards; and    -   wherein the normalization of lipid peroxide and pyroglutamic        acid levels in said body fluid samples correlates with the        synthesis or re-synthesis of glutathione in the patients        receiving IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™.

In a particular embodiment, the sample of body fluid is urine. Inanother particular embodiment, the sample of body fluid is whole blood.In a yet further particular embodiment, the sample of body fluid isplasma or serum.

In another preferred embodiment, if the levels of lipid peroxides,pyroglutamic acid, and glutathione are not normalized, the levels ofIMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ are increased indosage and the methods described above are repeated until suchnormalization occurs.

A fifth aspect of the invention provides a method for determining theamount of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ that isnecessary to reduce urine pyroglutamic acid in a subject in need of suchtherapy, comprising the steps of:

-   -   a) collecting a series of urine samples from a subject suspected        of being in need of such treatment, wherein said samples are        collected prior to the start of treatment, and daily after the        start of treatment for about 14 days;    -   b) measuring the amount of pyroglutamic acid in said samples;    -   c) comparing the amount of pyroglutamic acid in said samples        with that of a normal standard; and        wherein the reduction of pyroglutamic acid to normal levels in        said samples correlates with the amount of IMMUNE FORMULATION        100™ or IMMUNE FORMULATION 200™ sufficient to achieve a        beneficial effect.

In a preferred embodiment, if the levels of urine pyroglutamic acid arenot normalized, the levels of IMMUNE FORMULATION 100™ or IMMUNEFORMULATION 200™ are increased in dosage until such normalizationoccurs, and the methods described above are repeated until such timewhen normalization is achieved.

A sixth aspect of the invention provides a method for determining theamount of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ that isnecessary to reduce urine lipid peroxide in a subject in need of suchtherapy, comprising the steps of:

-   -   a) collecting a series of urine samples from a subject suspected        of being in need of such treatment, wherein said samples are        collected prior to the start of treatment, and daily after the        start of treatment for about 14 days;    -   b) measuring the amount of lipid peroxide in said samples;    -   c) comparing the amount of lipid peroxide in said samples with        that of a normal standard; and        wherein the reduction of lipid peroxide to normal levels in said        samples correlates with the amount of IMMUNE FORMULATION 100™ or        IMMUNE FORMULATION 200™ sufficient to achieve a beneficial        effect.

In a preferred embodiment, if the levels of urine lipid peroxide are notnormalized, the levels of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION200™ are increased in dosage until such normalization occurs, and themethods described above are repeated until such time when normalizationis achieved.

A seventh aspect of the invention provides a method for determining anorally anti-oxidative effective amount of IMMUNE FORMULATION 100™ orIMMUNE FORMULATION 200™ sufficient to diminish urine lipid peroxide andpyroglutamic acid levels and concurrently increase blood plasmaglutathione levels, comprising the steps of:

-   -   a) collecting blood plasma and urine samples prior to        administration of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION        200™ and daily after the start of administration for about 14        days;    -   b) measuring urine levels of lipid peroxide and pyroglutamic        acid;    -   c) measuring blood plasma glutathione levels;    -   d) determining whether a decrease in lipid peroxide and        pyroglutamic acid levels correlates with an increase in        glutathione levels; and        wherein said correlation establishes an orally anti-oxidative        effective amount of IMMUNE FORMULATION 100™ or IMMUNE        FORMULATION 200™.

In a preferred embodiment, the levels of lipid peroxide, pyroglutamicacid and glutathione are measured concurrently. In a further preferredembodiment, if the levels of lipid peroxides, pyroglutamic acid, andglutathione are not normalized when first tested, the levels of IMMUNEFORMULATION 100™ or IMMUNE FORMULATION 200™ are increased in dosage andthe methods described above are repeated until such normalizationoccurs.

An eighth aspect of the invention provides for establishing theinterdependence of lipid peroxides, pyroglutamic acid, glutathione, andimmune cell number and/or function in a subject suffering from oxidativestress, comprising the steps of:

-   -   a) collecting a urine sample from a subject suspected of being        under oxidative stress;    -   b) assaying the urine for the presence of lipid peroxides and        pyroglutamic acid;    -   c) collecting a sample of whole blood;    -   d) separating the cellular components from the liquid portion of        whole blood;    -   e) measuring glutathione in the liquid portion of whole blood        obtained in step d);    -   f) measuring the number of CD4+T cells and CD8+ T cells in the        cellular component of whole blood from step d); and    -   g) measuring the natural killer cell activity from the cellular        component of whole blood obtained from step d);        wherein a finding of decreased plasma glutathione levels, an        increase in urinary lipid peroxides and pyroglutamic acid, and a        decrease in the number of CD4+ and CD8+ T cells and natural        killer cell activity provides support for the interdependence of        the level of oxidative stress in said subject and immune cell        number and/or function.

A ninth aspect of the invention provides for a method for determining animmune enhancing effective amount of IMMUNE FORMULATION 100™ or IMMUNEFORMULATION 200™ sufficient to normalize CD4+, CD8+ T cell numbers andnatural killer cell activity in a subject suspected of experiencingoxidative stress, comprising the steps of:

-   -   a) collecting whole blood samples prior to administration of        IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ and daily        after the start of administration for about 14 days;    -   b) separating the cellular component of the whole blood from the        liquid component; and    -   c) measuring the number of CD4+ and CD8+ T cells and natural        killer cell activity using the cellular component from step b);        wherein a correlation between the dose of IMMUNE FORMULATION        100™ and IMMUNE FORMULATION 200™ that is sufficient to normalize        CD4+, CD8+ T cell numbers and natural killer cell activity        establishes an immune enhancing effective amount of IMMUNE        FORMULATION 100™ or IMMUNE FORMULATION 200™.

A tenth aspect of the invention provides for a method for determining anorally anti-oxidative effective amount and an immune enhancing effectiveamount of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ sufficientto normalize lipid peroxides, pyroglutamic acid and glutathione levelsin a subject suspected of experiencing oxidative stress, wherein saidnormalization of lipid peroxides, pyroglutamic acid and glutathionelevels results in immune enhancement, comprising the steps of:

-   -   a) collecting whole blood and urine samples prior to        administration of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION        200™ and daily after the start of administration for about 14        days;    -   b) measuring urine levels of lipid peroxide and pyroglutamic        acid;    -   c) separating the cellular component of the whole blood from the        liquid component;    -   d) measuring blood plasma glutathione levels using the liquid        component from step c);    -   e) measuring the number of CD4+ and CD8+ T cells and natural        killer cell activity using the cellular component from step c);    -   f) determining whether a decrease in urinary lipid peroxide and        pyroglutamic acid levels correlates with an increase in        glutathione levels, and whether the normalization of the levels        of all three of these products relates to a normalization of        CD4+ and CD8+ T cell numbers and normalization of natural killer        cell activity; and        wherein said correlation establishes an orally anti-oxidative        effective amount and an immune enhancing effective amount of        IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™.

In a particular embodiment, said correlation further establishes theinterrelationship of lipid peroxides, pyroglutamic acid, glutathione andimmune functions and the level of oxidative stress in said subject thatresults in depressed immune functions.

An eleventh aspect of the invention provides kits for measuringoxidative stress in an individual suspected of suffering from oxidativestress. Such a kit may contain all of the reagents necessary to measureat least three markers of oxidative stress at the same time. The assaykit may be formatted for use as a competitive or non-competitive ELISAassay. Alternatively, the kit may be structured in much the same way asa take home pregnancy kit, for example, using a test strip format. Thekit may also contain binding partners, for example, antibodies specificfor certain immune cells, such as CD4+ T cells, CD8+ T cells and naturalkiller cells. Thus, the kits of the present invention may be capable ofmonitoring both oxidative stress and immune cell number.

In a particular embodiment, the kit for measuring oxidative stress in asubject comprises:

-   -   a) a solid substrate containing immobilized binding partners        specific for at least three markers for oxidative stress;    -   b) either:        -   i) an enzyme conjugated second binding partner to the            oxidative stress markers; or        -   ii) a biotinylated second binding partner to the oxidative            stress markers;    -   c) either:        -   i) the enzyme substrate and the developing reagents specific            for the enzyme conjugated second binding partner from            step b) i); or        -   ii) a streptavidin conjugated third binding partner specific            for the second binding partner of step b) ii);    -   d) buffers for washing and sample dilution;    -   e) standards for each of the at least three markers of oxidative        stress; and    -   f) instructions for use of said kit.

In another particular embodiment, the binding partner is an antibody. Inanother particular embodiment the kit further comprises additionalcompartments to which have been attached antibodies specific for cellsurface markers for CD4+ T cells, CD8+ T cells and natural killer cells.In a preferred embodiment, the markers of oxidative stress are selectedfrom the group consisting of lipid peroxide, pyroglutamic acid andglutathione. In another preferred embodiment, the antibody is selectedfrom a monoclonal antibody, a polyclonal antibody, a chimeric antibody,and any combination thereof.

A twelfth aspect of the invention provides a method for providing acourse of therapy for an individual suspected or known to be sufferingfrom oxidative stress comprising

-   -   a) determining the identity and level of at least three markers        of oxidative stress in a sample of body fluid from said        individual, said markers being indicative of the extent of        oxidative stress; and    -   b) selecting the appropriate course of therapy for said        individual suffering from oxidative stress and the sequelae        thereof.

In a particular embodiment, the method includes administering saidappropriate course of therapy to said individual. In another particularembodiment, the method provides a course of therapy for an individualsuspected or known to be suffering from oxidative stress and monitoringthe success of said therapy comprising:

-   -   a) determining the identity and level of at least three markers        of oxidative stress in a sample of body fluid from said        individual, said marker being indicative of the extent of        oxidative stress;    -   b) selecting the appropriate course of therapy for said        individual suffering from said oxidative stress;    -   c) administering said appropriate course of therapy to said        individual; and monitoring the success of said therapy by        measuring a normalization in levels of said markers of oxidative        stress.

In a preferred embodiment, the method provides for said course oftherapy comprising administering IMMUNE FORMULATION 100™ or IMMUNEFORMULATION 200™ to said individual.

Other advantages of the present invention will become apparent from theensuing detailed description.

DETAILED DESCRIPTION

Before the present methods and treatment methodology are described, itis to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting, since the scope of the presentinvention will be limited only in the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein and/or which will become apparent to those persons skilled in theart upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the particular methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference.

Definitions

The terms used herein have the meanings recognized and known to those ofskill in the art; however, for convenience and completeness, particularterms and their meanings are set forth below.

“Treatment” refers to the administration of an antioxidant or theperformance of procedures with respect to a subject, for eitherprophylaxis (prevention) or to cure the infirmity or malady in theinstance where the subject is afflicted.

As used herein, “assessing the need for treatment” refers to determiningwhether a subject would be a candidate for therapy with an anti-oxidant.The determination would be made based on the particular disease andsymptoms associated with the disease, and whether or not the cause ofthe disease or condition can be attributed, at least in part, to highlevels of oxidation of cells, tissues, proteins or other molecular orchemical entities which are candidates for damage caused by oxidativestress, as evidenced by high levels of lipid peroxide and high levels ofPGA in the urine and low levels of glutathione in the blood.

A “therapeutically effective amount” is an amount sufficient to decreaseor prevent the symptoms associated with the disease caused by orattributed to oxidative stress.

By “patient” or “subject” is meant a human or non-human mammal that maybenefit from the therapies described in the present application, forexample, anti-oxidant therapy. The anti-oxidants may be administered tosubjects already having a disease or condition whose symptoms andsequelae are attributed to oxidation of proteins, cells or tissues, orparticular molecular entities or chemical compounds, and whose symptomsor sequelae may be alleviated by anti-oxidant therapy. Alternatively,the subjects may be predisposed to diseases or conditions caused by highlevels of oxidation, for which therapy with an anti-oxidant may bebeneficial. Accordingly, the subject may be treated prophylacticallywith the anti-oxidant therapy. Diseases or conditions for which suchanti-oxidant therapy would be beneficial. may be selected from the groupconsisting of autoimmune or degenerative diseases includingacetaminophen poisoning, ADD, Addison's disease, aging, AIDS, alopeciaareata, ALS, Alzheimer's disease, anemia (hemolytic), ankylosingspondylitis, arteriosclerosis, arthritis (including osteoarthritis andrheumatoid arthritis), asthma, autism, autoimmune disease, Behcet'sdisease, bums, cachexia, cancer, candida infection, cardiomyopathy(idiopathic), chronic fatigue syndrome, colitis, coronary arterydisease, cystic fibrosis, diabetes, Crohn's disease, eczema, emphysema,Epstein Barr Viral (EBV) syndrome, fibromyalgia, free radical overload,Goodpasture syndrome, Grave's disease, hepatic dysfunction (liverdisease), hepatitis B, hepatitis C, HIV or patients suffering from AIDS,hypercholesteremia (high blood cholesterol), herpes, infections (viral,bacterial and fungal), inflammatory bowel disease (IBD), lupus, maculardegeneration, malnutrition, Meniere's disease, multiple sclerosis,myasthenia gravis, neurodegenerative diseases, nutritional disorers,Parkinson's disease, Pemphigus vulgaris, Primary Billiary Cirrhosis,progeria, psoriasis, rheumatic fever, sarcoidusis, scleroderma,shingles, stroke, surgery, toxic poisoning, trauma, vasculitis,vitiligo, and Wegener'sgranulomatosis(nutritionadvisor.com/immunocalFAQ.html).

The term “utilization efficiency” as used herein refers to how well thebody uses the anti-oxidants which are administered to counteract thedamage caused by free radicals or other oxidizing agents which play arole in the damage to cells and tissues. The efficiency of use may bedetermined by either a direct measurement of the oxidized material, forexample, the levels of lipid peroxide and the levels of PGA in the urineor a specific oxidized protein such as oxidized low density lipoprotein(LDL), associated with cells or tissues, or found circulating in thebloodstream. The “utilization efficiency” is considered to be moreeffective when the level of oxidized material is decreased after therapywith an anti-oxidant compared to its level prior to the start of therapywith an anti-oxidant.

By “effectiveness of therapy” is meant that upon treating a subject withan anti-oxidant, one can determine whether the treatment has resulted inthe desired outcome. For example, in the case of treating a patienthaving high levels of an oxidized protein, for example, such as oxidizedLDL (low density lipoprotein), with an anti-oxidant, one may observe adecrease not only in the amount of oxidized LDL, but also in thesequelae associated with oxidized LDL, such as a decrease in the amountof atherosclerotic plaque which ultimately may lead to an increase orrisk of heart failure. In addition, patients suffering from HIV may alsohave sequelae that can be monitored after treatment with an antioxidant.These may include, for example, changes in the number of CD4+ T cellsand CD8+ T cells and their corresponding ratios.

“IMMUNE FORMULATION 100™” refers to a non-toxic nutritional compositionuseful for increasing glutathione production in a mammal in order toenhance the immune activity of the mammal. This composition contains thefollowing as essential active ingredients:

-   -   a: a catalytic quantity of elemental selenium or a water soluble        selenium precursor;    -   b: from about 5% to about 95% of a special whey product        containing from about 65% to about 85% protein which is from        about 65% to about 100% undenatured; and    -   c: from about 5% to about 95% by weight of colostrum;        the percent by weight of each component based on the total        weight of the composition. This material is further described        and claimed in U.S. Pat. No. 6,667,063.

“IMMUNE FORMULATION 200™” refers to a nutritional or therapeuticcomposition useful for treatment of mammals to enhance immune activity.This composition contains the following as essential active ingredients:a catalytic quantity of a selenium source together with glutathioneprecursors which are a mixture of glutamic acid, cystine or anotherrelated cystine precursor, and glycine in a molar ratio of about1:0.5:1, the amount of glutathione precursors being effective toincrease the content of glutathione in the body tissue of the mammalabove that of a pretreatment level thereby to enhance immune activity.This material is further described and claimed in U.S. Pat. No.6,592,908.

Glutathione is a tripeptide and a major reducing agent in the mammalianbody. Its chemical structure is:

or, more simply

GLU-CYS-GLY

Its chemical name is glutamyl-cysteinyl-glycine. Like many other smallpeptides in the mammalian body, it is not synthesized by proceduresinvolving DNA, RNA and ribosomes. Rather, it is synthesized from theamino acids available in the body and selenium by procedures utilizingenzymes and other body components such as adenosine triphosphate as anenergy source.

The term “anti-oxidative effective amount” as used herein refers to anamount of an anti-oxidant, such as for example, IMMUNE FORMULATION 100™or IMMUNE FORMULATION 200™ which, when delivered to a patient or subjectin need of such therapy results in reduction of the proteins, cellularcomponents, or tissue components that have been oxidized andsubsequently damaged or have reduced functional capacity as a result ofbeing oxidized. An “anti-oxidative effective amount” of an anti-oxidantis the amount of the anti-oxidant needed to restore certain functionalcapacities to the proteins, cells or tissues that are damaged byoxidation. The anti-oxidants are dietary supplements containingnutritional products. If an excess of any amino acid is used, it willpresumably be of nutritional value or may be metabolized.

“Pyroglutamic acid” or “PGA” is a keto derivative of proline that isformed nonenzymatically from glutamate, glutamine, andgamma-glutamylated peptides. It is also produced by the action ofgamma-glutamylcyclotransferase. It is also referred to as 5-oxoproline,5-pyrrolidone-2-carboxylic acid, or pyrrolidone-5-carboxylate. Elevatedlevels are often associated with problems of glutamine or glutathionemetabolism.

“Lipid peroxides” are fats that have been damaged by excess free radicalactivity. Lipid peroxides are the products of the chemical damage doneby oxygen free radicals to the lipid components of cell membranes. Thisoxidative damage, caused by free radical pathology, is thought to be abasic mechanism underlying many diverse pathologicalconditions—atherosclerosis, cancer, aging, rheumatic diseases, allergicinflammation, cardiac and cerebral ischemia, respiratory distresssyndrome, various liver disorders, irradiation and thermal injury, andtoxicity induced by certain metals, solvents, pesticides and drugs.Measurement of lipid peroxide levels plays a significant role inevaluating cellular damage caused by oxidative stress and determining anindividual's specific need for antioxidant supplementation. The level oflipid peroxides is an index of cellular membrane damage caused by theaction of free radicals. The organelle membranes, such as those of themitochondria, lysosomes, peroxisomes, and DNA can be damaged as well.This damage is lipid peroxidation, resulting from an excess ofprooxidants over antioxidants. Such excess, categorized as oxidativestress, can damage membrane proteins and cholesterol, as well asmembrane lipids. The elevation of lipid peroxides can serve as an earlywarning of the long-term effects of oxidative stress. The natural sequelof oxidative stress is chronic degenerative disease. One example is thatperoxidation of low density lipoproteins contributes to atherosclerosis.Other associated diseases include coronary artery disease and cancer,the leading causes of death in the United States.

The term “free radicals” refers to a chemical species that possesses anunpaired electron in the outer (valence) shell of the molecule. This isthe reason they are highly reactive and thus have low chemicalspecificity i.e., they can react with most molecules in their vicinity.This includes proteins, lipids, carbohydrates and DNA. Free radicalsattack the nearest stable molecule, thus “stealing” its electron. Whenthe attacked molecule loses its electron, it becomes a free radicalitself, thus beginning a chain reaction. It continues until the finalresult is the disruption of a living cell. Free radicals are producedcontinuously in cells either as by-products of metabolism ordeliberately as in phagocytosis (Cheeseman, K. H. and Slater, T. F., BrMed Bull. 1993 July; 49(3): 481-93).

“Surrogate biomarker” or “biomarker” or “marker” as used herein, refersto a highly specific molecule, the existence and levels of which arecausally connected to a complex biological process, and reliablycaptures the state of said process. Furthermore, a surrogate biomarker,to be of practical importance, must be present in samples that can beobtained from individuals without endangering their physical integrityor well-being, preferentially from biological fluids such as blood,plasma, urine, saliva or tears. The biomarkers of oxidative damage, asused herein, include increased lipid peroxides and pyroglutamic acid anddecreased glutathione. The levels of these biomarkers should reflect thedegree of oxidative stress in the body and are the result of certaindiseases or conditions and should continue to accumulate or remainstable in the body until released, excreted or neutralized. Furthermore,the presence of these biomarkers, in particular, lipid peroxides andpyroglutamic acid, should reflect the need for anti-oxidant therapy. Thenormalization of these biomarkers as well as normalization of the levelsof glutathione should also reflect the utilization efficiency andeffectiveness of anti-oxidative therapy as provided in the presentapplication.

A person “suspected of being in need of such treatment” in terms of themethods of the present invention may refer to an individual sufferingfrom symptoms suggestive of lowered immunity, such as frequentinfections or colds.

By the term “sequelae” of oxidative stress is meant the conditionsfollowing as a consequence of the level or duration of oxidative stress.This may include a predisposition for acquiring infections, or apredisposition to certain pathological conditions or diseases. It mayinclude cellular or tissue damage resulting from the persistence and/orbuildup of free radicals in particular tissues, for example, in kidneytissue following treatment with certain drugs such as adriamycin, orfollowing an injury to the brain or spinal cord.

The term “antibody” as used herein includes intact molecules as well asfragments thereof, such as Fab and F(ab′)₂, which are capable of bindingthe epitopic determinant. Antibodies that bind the proteins or themarkers of oxidative stress of the present invention can be preparedusing intact polypeptides or fragments containing small peptides ofinterest as the immunizing antigen attached to a carrier molecule.Commonly used carriers that are chemically coupled to peptides includebovine or chicken serum albumin, thyroglobulin, and other carriers knownto those skilled in the art. The coupled peptide is then used toimmunize the animal (e.g, a mouse, rat or rabbit). The antibody may be a“chimeric antibody”, which refers to a molecule in which differentportions are derived from different animal species, such as those havinga human immunoglobulin constant region and a variable region derivedfrom a murine mAb. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567;and Boss et al., U.S. Pat. No. 4,816,397.). The antibody may be a humanor a humanized antibody. The antibody may be a single chain antibody.(See, e.g., Curiel et al., U.S. Pat. No. 5,910,486 and U.S. Pat. No.6,028,059). The various portions of the chimerized antibodies can bejoined together chemically by conventional techniques, or can beprepared as a contiguous protein using genetic engineering techniques.For example, nucleic acids encoding a chimeric or humanized chain can beexpressed to produce a contiguous protein. See, e.g., Cabilly et al,U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European PatentNo. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S.et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat.No.5,225,539; Winter, European Patent No. 0,239,400 B1; and Queen etal.. U.S. Pat. Nos. 5,585,089, 5,698,761 and 5,698,762. See also,Newman, R. et al., BioTechnology, 10: 1455-1460 (1992), regardingprimatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 andBird, R. E. et al., Science, 242: 423-426 (1988)) regarding single chainantibodies. The antibody may be prepared in, but not limited to, mice,rats, rabbits, goats, sheep, swine, dogs, cats, or horses.

General Description

Finding a means of protecting cells and/or molecules from the effects offree radicals is of obvious medical importance. These free radicals havebeen associated with a large variety of conditions in which cells die,resulting in severe clinical consequences. Furthermore, the need forprotection of cells and/or molecules from the damage caused by freeradicals has been addressed, in part, by administration ofanti-oxidants. However, it has been difficult to measure the presence offree radicals due to their very short half-life. Furthermore, themethods that are available, such as electron spin resonance and spintrapping methods (Cheeseman, K.H. and Slater, T. F., Br Med Bull..1993July; 49(3): 481-93) using exogenous compounds having a high affinityfor free radicals, have poor sensitivity and are not 100% accurate, thatis, they produce only semi-quantitative data.

Therefore, it is necessary to find alternate strategies to assess thepresence of free radicals and the level of oxidative stress in anindividual patient. A commonly used technique is to measure a marker offree radicals rather than the radical itself (Slater, T. F., MethodsEnzymol. 1984; 105:283-93; Pryor W. A. and Godber S. S., Free Radic BiolMed. 1991; 10(3-4):173). Thus, various assays have been devised tomeasure these markers of oxidative stress.

Likewise, it is difficult to assess whether an individual is in need oftreatment with an anti-oxidant. Furthermore, once it has been determinedthat an individual would benefit from such therapy, it is important tobe able to assess whether the therapy as delivered is being utilized toits full capacity. And finally, it is necessary to determine howeffective that therapy is by assessing an individual's response andoutcome following such therapy.

Accordingly, the present invention provides a multidimensional andcomprehensive method for assessing the need for treatment of a subjectwith an anti-oxidant. Previous tests for measuring an individual's levelof oxidative stress has relied primarily on the measurement of one ortwo markers of oxidative stress, such as lipid peroxides. The presentinvention provides for the quantitation of several markers of oxidativestress, including lipid peroxides, pyroglutamic acid, and glutathione.In addition, since it is becoming more apparent that an individual'simmune status may depend in part on the level of glutathione present invivo, the present invention provides for concurrent measurement ofimmune cell numbers and activity, in particular, the number of CD4+ andCD8+ T cells, and the level of natural killer cell activity. The presentinvention will thus provide for the interrelationship between lipidperoxides, pyroglutamic acid, and glutathione, as well as the immunefunction's associated with adequate levels of antioxidants, and themarkers thereof. There have been no known studies of which the inventoris aware that provides this comprehensive and quantitative manner ofassessment of oxidative stress and related immune functions. It is afurther object of the present invention to be able to measure theutilization efficiency of the anti-oxidant therapy once an individualhas started therapy with an anti-oxidant. It is yet a further object ofthe present invention to be able to measure the effectiveness of theanti-oxidant therapy once an individual has started and then completedthe therapy with an anti-oxidant.

Furthermore, this invention is based, in part, on the use of novelIMMUNE FORMULATIONs, in particular, IMMUNE FORMULATION 100™ or IMMUNEFORMULATION 200™, and provides for methods of determining the amount ofthese formulations necessary to increase glutathione synthesis orre-synthesis in a patient in need of such therapy and to reduce theamounts of lipid peroxides and pyroglutamic acid levels in thesepatients. The ability to monitor the elevation in plasma glutathionelevels while concurrently measuring a decrease in two oxyradicalmetabolites, urine lipid peroxide and pyroglutamic acid, provides a morequantitative and accurate assessment of a patient's level of oxidativestress, and allows for adjustment in dose and duration of appropriateanti-oxidant therapies, including those described herein.

Accordingly, the present invention provides for tracking two metabolitesfrom their respective metabolic pathways to establish anti-oxidant needbefore treatment with specific anti-oxidant formulations, and after theadministration of the anti-oxidants to demonstrate the utilizationefficiency of the therapy and eventual effectiveness of the therapy onglutathione synthesis. In preferred embodiments, the anti-oxidanttherapies are IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™.

Thus, it is important to track the oxyradical metabolite urine lipidperoxide before and after therapy to determine where the level falls ascompared to the normal range. The normal range as determined by standardclinical testing, such as that provided by Metametrix ClinicalLaboratory, is about 8.9 to 13.3 nM/mg.

Furthermore, it is also crucial to track the catabolic breakdown productof glutathione, known as pyroglutamic acid (PGA) in the urine and todetermine where the level falls as compared to the normal range. Thenormal range as determined by standard clinical testing, such as thatprovided by Metametrix Clinical Laboratory, is about <80 μg/mg ofcreatinine.

Therefore, establishing the patient's baseline level of thesemetabolites will help to establish whether there is a need foranti-oxidant therapy. If the level falls outside of the normal range forthese metabolites, there is a need for initiation of anti-oxidanttherapy.

Thus, once certain anti-oxidant deficiencies have been established whichmay be responsive to therapies with anti-oxidants, it would beimperative to determine the utilization efficiency of such therapy onceit has been initiated. Therapies such as IMMUNE FORMULATION 100™ orIMMUNE FORMULATION 200™ would in all likelihood be most beneficial, dueto the effects of these formulations on production of glutathione andits anti-oxidant activity per se and/or its antioxidant activity withselenium through glutathione peroxidase. Afterwards, the overalleffectiveness of such therapies must be monitored such as by the methodsof the present invention to determine whether normal levels of lipidperoxides, PGA and glutathione have been established. The determinationas to whether there is a need for continued therapy would be made by thehealth care assistant, physician, or by self-assessment.

Methods of Quantifying Lipid Peroxides

When a fatty acid is peroxidized it is broken down into aldehydes, whichare excreted. Aldehydes such as thiobarbituric acid reacting substances(TMARS) have been widely accepted as a general marker of free radicalproduction, with the most commonly measured TBARS being malondialdehyde(Esterbauer, H, Jürgens, G, Quehenberger, O, & Koller, E, J Lipid Res1987, 28:495-509; Buege, J A & Aust, S D, Methods Enzymol 1978,52:302=310; Wallin, B & Camejo, G , Scand J Clin Lab Invest 1994,54:341-346; El-Saadani, M, Esterbauer, H, El-Sayed, M, Goher, M, Nassar,A Y, & Jiirgens, G A, J Lipid Res 1989, 30:627-630; Esterbauer, H,Gebicki, J, Puhl, H, & Jurgens, G, Free Radic Biol Med 1992,13:341-390).

Methods of measuring oxidized lipids are well known to those of skill inthe art (see, e.g., Vigo-Pelfrey et al. Membrane Lipid Oxidation, VolumeI-II. CRC Press). such methods include, but are not limited to massspectrometry, absorption spectrometry (e.g., using UV absorbance at 234nm), liquid chromatography, thin layer chromatography, and the use ofvarious “oxidation-state” sensitive reagents, e.g. in various redoxreactions.

Previously known methods for measuring oxidized lipids (e.g. lipidperoxides), include the Wheeler method, iron thiocyanate method,thiobarbituric acid method, and others. The Wheeler method (Wheeler(1932) Oil and Soap, 9: 8997) is that in which oxidized lipid is reactedwith potassium iodide to isolate iodine, which is then titrated with asodium thiosulfate standard solution. In the iron thiocyanate method(Stine et al. (1954) J. Dairy Sci., 37: 202) oxidized lipid peroxide ismixed with ammonium thiocyanate and ferrous chloride, and the blue colorfrom the resulting iron thiocyanate is calorimetrically determined. Inthe thiobarbituric acid method (Tappel and Zalkin (1959) Arch. Biochem.Biophys., 80: 326) the lipid peroxide is heated under acidic conditionsand the resulting malondialdehyde is condensed with thiobarbituric acidto form a red color dye, which is then calorimetrically measured.

In another approach, it has been demonstrated that peroxidase decomposeslipid peroxides and that the resulting reaction system colors intenselywith increasing quantities of lipid peroxide, if an adequate hydrogendonor is present in the reaction system (see, e.g., U.S. Pat. No.4,367,285) Thus, in one embodiment, the assays of this invention mayutilize a peroxidase and a hydrogen donor.

Many peroxidases are suitable. The peroxidase employed in the presentinvention is preferably any of the commercially available horseradishperoxidases.

The hydrogen donor employed may be any of the known oxidizable compoundswhich, preferably, generate color, fluorescence or luminescence uponoxidation. The conventional coloring, fluorescent, luminescent reagentsmay be utilized. The known coloring reagents which may be employedinclude, but are not limited to guaiacol, 4-aminoantipyrine with phenol,4-aminoantipyrine with N,N-dimethylaniline, 3-methyl-2-benzothiazolinonewith dimethylaniline, ortho-dianisidine, and the like. Typically usefulfluorescent reagents include, but are not limited to homovanillic acid,p-hydroxyphenylacetic acid, and the like. Suitable luminescent reagentsinclude but are not limited to luminol and the like. The amount of thehydrogen donor employed is preferably at least equimolar, preferably notless than two moles, per mole of lipid peroxide contained in testsample. The amount may be varied depending upon the size of the sampleand the content of the lipid peroxide in the sample.

Suitable reaction mediums which may be employed include, but are notlimited to dimethylglutarate-sodium hydroxide buffer solution, phosphatebuffer solution and, Tris-hydrochloric acid buffer solution is normallyfrom about pH 5 to about pH 9.

Such factors as the pH at the time of reaction, the reaction period, themeasuring wavelength, etc., may be varied depending upon the reagentsemployed. Suitable conditions can be selected according to thecircumstances.

Another class of assays for oxidized lipids is described in U.S. Pat.No. 4,900,680. In this approach, an oxidized lipid (e.g. ahydroperoxide) is reacted with a salt or hydroxide of a transition metalwhich produces a cation having a valency of 2, a complex of a transitionmetal having a valency of 2, a heme, a heme peptide, a heme protein, ora heme enzyme. The resultant active oxygen and oxygen radicals reactwith a luminescent substance, and light emitted by this reaction isoptically measured. Examples of a catalyst acting on a lipidhydroperoxide to produce active oxygen species such as active oxygen oroxygen radicals are: a transition metal salt which produces a cationhaving a valency of 2 (e.g., ferrous chloride, ferrous sulfate,potassium ferricyanide, each of which produces Fe²⁺; manganous chlorideor manganous sulfate, each of which produces Mn²⁺; or cobalt chloride orcobalt sulfate, each of which produces Co²⁺); a hydroxide of thetransition metals described above; a complex of a transition metalhaving a valency of 2 (e.g., Fell -porphyrin complex); a heme protein(e.g., cytochrome C, hemoglobin, or myoglobin); a. heme peptide (e.g., acompound obtained by decomposing a heme protein by a protease such aschymotrypsin or trypsin); and a heme enzyme (e.g., horseradishperoxidase or prostaglandin peroxidase).

Particular catalyst compounds include, but are not limited to, a hemeprotein, a heme peptide, or a heme enzyme. Most usually, the hemeprotein such as cytochrome C is used due to easy handling. Theconcentration of the catalyst compound preferably ranges from about 0.1μg/ml to about 1,000 μg/ml and usually falls within the range of about 1μg/ml to about 200 μg/ml. For example, best luminous efficiency can beobtained when the concentration is about 10 μg/ml for cytochrome C,about 120 μg/ml for cytochrome C heme peptide; and about 10 μg/ml forhorseradish peroxidase.

The luminescent substance is not limited to a specific one, provided itreacts with active oxygen or an oxygen radical to emit light. Examplesof such a compound include, but are not limited to polyhydroxyphenols(e.g., pyrogallol, perprogalline etc.), phthaladine derivatives (e.g.,luminol, isoluminol, etc.), indol derivatives (e.g., indoleacetic acid,skatole, tryptophan, etc.); thiazolidine derivatives (e.g., Cypridinacealuciferin, lophine, etc.), an acrydine derivatives (e.g., lucigenine),oxalic acid derivatives (e.g., bistrichlorophenyloxalate); and1,2-dioxa-4,5-azine derivatives. The concentration of the luminescentsubstance varies depending on the compound used. The concentration ispreferably 0.1 μg/ml or more. When luminol is used, its concentration ismost preferably 1 μg/ml.

Measurements are preferably performed in a weak basic solution of aluminescent reagent such as a heme protein and luminol. A particular pHvalue ranges from about pH 9 to about pH 10. Many buffers are suitable.One particular buffer is a borate buffer (H₃ BO₃—KOH), a carbonatebuffer (Na₂CO₃—NaHCO₃), a glycine buffer (NH₂CH₂COOH—NaOH), or the like.The borate buffer is most preferred.

In order to prevent oxygen dissolved in the luminescent reagent solutionfrom interfering with the analysis of a very small amount of oxidizedlipid, the luminescent reagent solution is desirably purged with aninert gas to remove oxygen to obtain a stable measurement value.Examples of the inert gas are nitrogen gas and argon gas.

The concentration of the oxidized lipid in the sample is calculatedbased on a calibration curve. The calibration curve can be formedaccording to standard methods, e.g., by using a material selected frommethyl linolate hydroperoxide, arachidonic acid hydroperoxide,phosphatidylcholine hydroperoxide, phosphatidylethanolaminehydroperoxide, and triacylglycerol hydroperoxide.

The methods of this invention may utilize fluorescent materials whosefluorescence is altered by oxidation state. Such fluorescent materialsare well known to those of skill in the art and include, but are notlimited to 2′7′-dichlorodihydrofluorescine diacetate, rhodaminecis-parinaric acid, NBD, cis-parinimic acid cholesteryl esters,diphenylhexatriene proprionic acid, and the like. The use of suchindicators is illustrated in the examples.

It will be appreciated that the foregoing methods ofdetecting/quantifying oxidized lipids are intended to be illustrativeand not limiting. Numerous other methods of assaying oxidized lipids areknown to those of skill in the art and are within the purview of thisapplication.

The TBA test has been challenged because of its lack of specificity,sensitivity, and reproducibility. The use of liquid chromatographyinstead spectrophotometer techniques help reduce these errors. Inaddition, the test seems to work best when applied to membrane systemssuch as microsomes. Gases such as pentane and ethane are also created aslipid peroxidation occurs. These gases are expired and commonly measuredduring free radical research. Dillard (Dillard et al. Free Radic BiolMed. 1989; 7(2):193-6) was one of the first to determine that expiredpentane increased as VO2 max increased. Kanter et al. (Kanter M M, NolteL A, Holloszy J O, J Appl Physiol. 1993 February; 74(2):965-9) hasreported that serum MDA levels correlated closely with blood levels ofcreatine kinase, an indicator of muscle damage. Lastly, conjugateddienes (CD) are often measured as indicators of free radical production.Oxidation of unsaturated fatty acids results in the formation of CD. TheCD formed are measured and provide a marker of the early stages of lipidperoxidation (Poirier B. Michel O, Bazin R, Bariety J, Chevalier J,Myara I. Nephrol Dial Transplant. 2001 August; 16(8):1598-606). A newlydeveloped technique for measuring free radical production shows promisein producing more valid results. The technique uses monoclonalantibodies and may prove to be the most accurate measurement of freeradicals. However, until further more reliable techniques areestablished it is generally accepted that two or more assays be utilizedwhenever possible to enhance validity.

When glutathione or glutathione peroxidase (which uses selenium) or ADPfor regeneration of oxidized glutathione is deficient, blood lipidperoxide and subsequently urine lipid peroxide would increase.

Oral ingestion of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ canreplenish glutathione levels and have a vital antioxidant effect, thus,reducing blood lipid peroxide and consequently urine lipid peroxide.

This metabolite, urine lipid peroxide, can mirror the body's antioxidantneed and mirror the utilization efficiency of IMMUNE FORMULATION 100™ orIMMUNE FORMULATION 200™ to remediate that deficiency. It can also mirrorthe effectiveness of glutathione synthesis from IMMUNE FORMULATION 100or IMMUNE FORMULATION 200™ during that deficiency. However, the amountof IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ needed to reversethe deficiency is to be quantified so as to establish a cause and effectinterrelationship.

The following equation demonstrates that a toxic peroxide can bedetoxified by glutathione to form water and oxidized G—S—S-G.

${{2G\text{-}{SH}} + {H_{2}O_{2}}}\underset{({{with}\mspace{14mu} {selenium}})}{\overset{\mspace{25mu} {{glutathione}\mspace{20mu} {peroxidase}}\mspace{34mu}}{\rightarrow}}{{G\text{-}S\text{-}S\text{-}G} + {2\; H_{2}O}}$

The high level of toxic lipid peroxide in the urine would indicate thatsufficient glutathione or selenium is not available to detoxify thatperoxide. This can be tested and proven by quantifying the collaborationbetween the oral ingestion of specified amounts of IMMUNE FORMULATION100™ or IMMUNE FORMULATION 200™ and the reduction of lipid peroxide inthe urine. Concurrent measurement of plasma glutathione levels would bemade to confirm that the therapy resulted in synthesis or resynthesis ofglutathione levels. The normal range for glutathione in the plasma isabout 200-400 mole/L (AmScotm, Cincinnati, Ohio).

Therefore, oral ingestion of an antioxidant such as IMMUNE FORMULATION100™ or IMMUNE FORMULATION 200™ should result in reduced urinaryexcretion of lipid peroxide and increased levels of glutathionesynthesis in blood plasma, thus proving that these three functions areinterrelated and interdependent.

There are several methods and kits available and known to those skilledin the art for measuring the presence of lipid peroxide in bodilyfluids, including urine. Using these methods, the normal range of urinelipid peroxide is about 8.9 to 13.3 nM/mg creatinine (MetametrixClinical Laboratory, Norcross, Ga.). Using this as the normal range, thephysician assessing his or her patient for the level of oxidative stresscan use one of the validated means for measuring urine lipid peroxideand obtain a value for that patient to determine where he or she is inrelation to this normal value. If the level increases to a level outsideof the normal range, he or she is in need of therapy with ananti-oxidant. In one particular embodiment, a patient in need ofanti-oxidant therapy has a urine level of lipid peroxide which is abovethe normal range. Preferably, the anti-oxidant of choice is IMMUNEFORMULATION 100™ or 200™, although other anti-oxidant therapies are alsocontemplated for use with the invention.

Furthermore, upon initiation of therapy with an anti-oxidant, it is ofuse to determine the utilization efficiency of such therapy bymonitoring changes in urine lipid peroxide, to determine if the level isbeing adjusted to within the normal range. In addition, these levelswould be monitored throughout the course of therapy to determine theeffectiveness of therapy with the anti-oxidant. The measurement of urinelipid peroxide may be done prior to, concurrently with, or shortlythereafter, a measure of urine pyroglutamic acid and plasma glutathionelevels in order to provide a secondary confirmation and assessment ofthe patient's oxidative stress level. Furthermore, these tests may becombined with other test parameters to determine whether the patient hasrecovered sufficiently in order for therapy with the anti-oxidant to bediscontinued. For example, in the case of a patient suffering fromdiabetes or high cholesterol or high triglyceride levels, the physicianmay assess other parameters such as glycated hemoglobin, glucose levelsor request a full battery of blood chemistries to assess the overallhealth status of the individual before discontinuing therapy with theanti-oxidant.

The methods for measuring lipid peroxidation products and lipidperoxidation damage in tissues, cells and body fluids have beendescribed above. The choice of which method is most appropriate depends,among other things, on whether the measurement is needed for strictlyresearch purposes, or for a particular medical situation or conditionwherein the patient is under the care of a qualified physician, orhealth care worker, or uses a self-administered urine kit. In theroutine clinical research laboratory, the determination ofthiobarbituric acid reactive substances (TBARS) under strictlystandardized conditions is in most cases the first choice. Thespecificity of the colorimetric or fluorimetric assay can besignificantly improved if it is combined with analysis by HPLC methods.If the level of TBARS is increased, other more sophisticated assaysshould be performed for verification or validation of the valuesobtained. Several such assays are available including: Phospholipid- andcholesterylester hydroperoxides, aldehydic lipid peroxidation productsincluding 4-hydroxynonenal, fluorescent protein adducts (e.g.lipofuscin), conjugated dienes and antioxidants. The measurement ofpentane and ethane in the exhaled air by gas chromatography has been theonly available non-invasive method, although this assay method has itsown drawbacks in that it is time consuming and cumbersome to runroutinely. Several laboratories have also developed immunological assayssuch as the enzyme-linked immunosorbent assay (ELISA) or theradioimmunoassay (RIA) for determining proteins modified by lipidperoxidation products (e.g. malondialdehyde, 4-hydroxynonenal) orautoantibodies against oxidatively modified proteins. These assaysprovide a much more convenient and quantitative assessment of theby-products of oxidation and are much less cumbersome and time consumingto run as compared to the HPLC or other standard chemical approaches forassay and quantitation.

Under standard laboratory practice, and for assessment of a patient'sclinical condition, an increased concentration of end products of lipidperoxidation is the evidence most frequently cited for the involvementof free radicals in human disease. However, while it is recognized thatthe generation of free radicals and the subsequent oxidative damageresulting from their presence occurs in most diseases, it is also truethat oxidative damage plays a significant pathological role in only someof these diseases. This is true, for example, in atherosclerosis and inexacerbation of the initial tissue injury caused by ischemic ortraumatic brain injury or spinal cord injury, where peroxidation of thesurrounding cells and tissues appears to be extremely important in thepathology of these conditions. Moreover, it is also becoming moreapparent that stress plays a role in reduction in immune responsivenessto known pathogens and that this may be due in part to reduced levels ofglutathione. (Leonore A. Herzenberg et al, Proc. Natl. Acad. Sci. USAVol. 94, pp. 1967-1972, March 1997) Oxidative stress can damage manybiological molecules including proteins, DNA and lipids. Many assays areavailable to measure lipid peroxidation, but no single assay is anaccurate measure of the whole process. Application of simplediene-conjugate and thiobarbituric acid (TBA) assays to human tissuesand body fluids can produce artifacts. Thus, it is important to utilizeother methods for assessment of a more accurate clinical profile.

Some of the methods used to monitor oxidative products include thefollowing:

Determination of Urinary Thiobarbituric-Acid-Reacting Substances (TBARS)This procedure involves the incubation of a sample of urine with 5%butylated hydroxytoluene (in glacial acetic acid) and 0.5% aqueousthiobarbituris acid (TBA) solution (Buege J A, Aust S D: Microsomallipid peroxidation. Methods Enzymol (1978), 52:302-310; Valenzuela A:The biological significance of malondialdehyde determination in theassessment of tissue oxidative stress. Life Sci 1991, 48:301-309). Aftermixing and incubating the mixture for about 30 minutes, the absorbanceis measured at 532 nm using a spectrophotometer. The quantity of TBARSis proportional to the amount of malondialdehyde (MDA), a lipidperoxidation product generated by the oxidation of lipids by reactiveoxygen spoecies. MDA reacts with TBA to form a 1:2 MDA:TBA adduct thatabsorbs at 532 nm. To control for urine concentration, data arenormalized to urine creatinine concentrations as described by Coulthardet al. (Coulthard M G, Hey E N, Ruddock V: Creatinine and ureaclearances compared to inulin clearance in preterm and mature babies.Early Hum Dev 1985, 11:11-19).

Measurement of 8-epi-prostaglandin PGF2a (8-epi-PGF2a) The appearance of8-epi-prostaglandin PGF2a (8-epi-PGF2a) in plasma or urine has beensuggested by a number of investigators as a reliable index of in vivofree radical generation and oxidative lipid formation. There is verystrong evidence from animal studies that 8-epi-PGF2a increase in plasmaand urine as a result of oxidative stress, and in human, this product iselevated in smokers. Comparison with other measures of lipidperoxidation, 8-epi-PGF2a is specific product of lipid peroxidation, andis very stable. In addition, its formation is modulated by antioxidantstatus, and its level is not affected by lipid content of the diet. Themeasurement of 8-epi-prostaglandin PGF2 can be done using a standardimmunoassay using antibodies specific for this by-product of lipidoxidation.

ELISA kits for measurement of 8-hydroxy-2’-deoxyguanosine (8-OHdG)8-OhdG is a nucleotide which is excised from DNA. Endonuclease repairenzymes work quickly therefore the amount excised in urine directlyreflects a person's degree of damage in the body. These kits, which areavailable from Genox, measure the amount of 8-hydroxy-2′-deoxyguanosine(8-OHdG) in urine, serum, plasma, tissue homogenate and digestedlymphocyte DNA samples. Genox is a distributor of products developed bythe Japan Institute for the Control of Aging. Furthermore, Genox alsosells a monoclonal antibody against 8-OhdG for immunohistochemicalstudies on tissue samples. These kits contain the following.

  8-OHdG coated microtiter plate (split type) Primary antibody(Anti-8-OHdG monoclonal antibody) Primary antibody solution Secondaryantibody (POD-conjugated anti mouse antibody) Secondary antibodysolution Chromatic solution (3,3′,5,5′-tetramethylbenzidine) Substratesolution Washing solution Reaction terminating solution Standard 8-OHdGsolution (0.5, 2, 8, 20, 80, 200 ng/ml Plate seal

The general procedure is as follows:

-   -   (1) Primary Antibody Reaction (Competitive Reaction): 37° C. for        lhour    -   (2) Secondary Antibody Reaction: 37° C. for ihour    -   (3)Development of Color Reaction: Room Temperature for 15min in        the dark    -   (4) Absorbance Reading (wavelength at 450 nm) and Calculation of        Results

Additional methods for measuring 8-hydroxy-2′-deoxyguanosine (8-OHdG)are essentially as outlined in the following references, which areincorporated in their entirety. (S. S. Kantha, S. Wada, H. Tanaka, M.Takeuchi, S. Watabe, and H. Ochi Carnosine sustains the retention ofcell morphology in continuous fibroblast culture subjected tonutritional insult. Biochemical and Biophysical Research Communications,223, 278-282 (1996); S. S. Kantha, S. Wada, M. Takeuchi, S. Watabe, andH. Ochi, A sensitive method to screen for hydroxyl radical scavengingactivity in natural food extracts using competitive inhibition ELISA for8-hydroxydeoxyguanosine; Biotechnology Techniques, 10(12), 933-936(1996); J. Leinonen, T. Lehtimaki, S. Toyokuni, K. Okada, T. Tanaka, H.Hiai, H. Ochi, P. Laippala, V. Rantalaiho, O. Wirta, A.Pastemack, andH.Alho, New biomarker evidence of oxidative DNA damage in patients withnon-insulin-dependent diabetes mellitus; FEBS Letters, 417, 150-152(1997); H. Tsuboi, K. Kouda, H.T akeuchi, M. Takigawa, Y. Masamoto, M.Takeuchi, and H.Ochi, 8-Hydroxydeoxyguanosine in urine as an index ofoxidative damage to DNA in the evaluation of atopic dermatitis, BritishJournal of Dermatology, 138, 1033-1035 (1998); Y. Miyake, K. Yamamoto,N. Tsujihara, and T. Osawa Protective effects of lemon flavonoids onoxidative stress in diabetic rats. Lipids, 33(7), 689-695 (1998); M-H.Kang, M. Naito, N. Tsujihara, and T. Osawa Sesarnolin inhibits lipidperoxidation in rat liver and kidney. Journal of Nutrition, 128,1018-1022 (1998); T. Arimoto, T. Yoshikawa, H. Takano, M. KohnoGeneration of reactive oxygen species and 8-hydroxy-2′-deoxyguanosineformation from diesel exhaust particles components in L1210 cells.Japanese Journal of Pharmacology, 80, 49-54 (1999) ; M. D. Evans, M. S.Cooke, I. D. Podmore, Q. Zheng, K. E. Herbert, and J. Lunec,Discrepancies in the measurement of UVC-induced 8-oxo-2′-deoxyguanosine:Implications for the analysis of oxidative DNA damage. Biochemical andBiophysical Research Communications, 259, 374-378 (1999)).

AntiOxidant Check by Body Balance is a safe, easy-to-use, and reliabletest that uses a small urine sample to measure free radical activity bymeasuring lipid peroxide levels. The kit provides a sample collectiondevice for urine, which is collected and forwarded to a laboratory foranalysis.

Oxis Bioxytech LP0586 from Oxis International (Seattle, Wash.) is acolorimetric assay for evaluating lipid peroxidation. The results arespecific for malondialdehyde and 4-hydroxyalkenals, which are markers oflipid peroxidation.

Methods for Measuring Glutathione

Glutathione is recycled and reutilized in the kidney, after its threeconstituent amino acids are broken down in the renal tubules. Any aminoacid that is not considered healthy or optimal is theoretically excretedvia the renal tubules, and glutathione cannot be resynthesized unless itgains an optimal amount of its three precursor amino acids, cysteine,glycine and glutamate. When there is a deficiency of one of these aminoacids, especially cysteine, glutathione cannot optimally beresynthesized. It is thus broken down and excreted in the urine via itsconstituent parts as waste products, one of which is pyroglutamic acid,described below. PGA is a crucial marker of glutathione breakdown andits deficient reutilization in the kidney. Therefore, it is crucial toestablish the level of this depletion and to determine whether IMMUNEFORMULATION 100™ or IMMUNE FORMULATION 200™ can replenish glutathionelevels and reverse the excessive excretion of urine lipid peroxide. Itwould be possible to use urine PGA as a marker of glutathione need,glutathione utilization, and glutathione resynthesis and of IMMUNEFORMULATION 100™ or IMMUNE FORMULATION 200™ efficiency. Thus, high urinePGA is a marker for glutathione need, diminished glutathione utilizationand deficient glutathione resynthesis.

Several methods are currently available for measuring glutathionelevels. The normal level of plasma glutathione as measured by AmScot™ inCincinnati, Ohio is about 200-400 mole/L. The methods for measurement ofglutathione are listed below.

The Bioxytech GSH-400 kit from Oxis International (Seattle, Wash.) is anon-enzymatic, colorimetric assay specific for glutathione.

Cayman Chemical produces a glutathione assay kit, which utilizes acarefully optimized enzymatic recycling method, using glutathionereductase for the quantification of GSH. (Baker, M. A., Cerniglia, G.J., and Zaman, A. Microtiter plate assay for the measurement ofglutathione (GSH) and glutathione disulfide (GSSG) in large numbers ofbiological samples. Anal. Biochem. 190, 360-365 (1990); Eyer, P. andPodhradsky, D. Evaluation of the micromethod for determination ofglutathione using enzymatic cycling and Ellman's reagent. Anal. Biochem.153, 57-66 (1986); Tietze, F. Enzymic method for quantitativedetermination of nanogram amounts of total and oxidized glutathione:Applications to mammalian blood and other tissues. Anal. Biochem. 27,502-522 (1969)). Briefly, the sulfhydryl group of GSH reacts with DTNB(5,5′-dithio-bis-2-nitrobenzoic acid, Ellman's reagent) and produces ayellow colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide,GSTNB (between GSH and TNB) that is concomitantly produced, is reducedby glutathione reductase to recycle the GSH and produce more TNB. Therate of TNB production is directly proportional to this recyclingreaction, which in turn is directly proportional to the concentration ofGSH in the sample. Measurement of the absorbance of TNB at 405 or 412 nmprovides an accurate estimation of GSH in the sample. GSH is easilyoxidized to the disulfide dimer GSSG. Because of the use of glutathionereductase in the Cayman GSH assay kit, both GSH and GSSG are measuredand the assay reflects total glutathione. The kit can also be used tomeasure only GSSG by following an alternative protocol. GSH measurementcan be done in plasma, tissue samples, and cultured cells using thiskit. Nearly all samples require deproteination before assay.

Methods for Measuring Pyroglutamic Acid

The presence of pyroglutamic acid (PGA), also known as 5-oxoproline, inthe urine is an indication of a defect in the γ-glutamyl cycle, a seriesof enzyme-linked reactions involved in the synthesis, metabolism, andtranscellular transport of glutathione. Furthermore, it is present inurine in patients suffering from diseases or conditions which inducehigh levels of oxidative stress, such as those conditions outlined inthe present application. In these conditions, the presence of abnormallyhigh levels of PGA is an indication that glutathione levels are low andthat there is a defect in glutathione re-synthesis. The normallaboratory range for PGA is <80 μg/mg of creatinine (MetaMetrix ClinicalLaboratory, Norcross, Ga.). The levels of creatinine are measured usingstandard clinical laboratory procedures known to those skilled in theart.

One object of the present invention is to determine if and how muchIMMUNE FORMULATION 100™ or 200™ would diminish PGA excretion in theurine and how much IMMUNE FORMULATION 100™ or 200™ would be needed toincrease glutathione synthesis or resynthesis during this deficiency.Glutathione levels would be tested in blood plasma while PGA would betested concurrently in the urine. Most preferably, boih urine lipidperoxides and PGA would both be measured concurrently with plasmaglutathione to aid in accuracy of the test results and to establish amore precise assessment of the need for treatment with an antioxidant,such as IMMUNE FORMULATION 100™ or 200™. Therefore, oral ingestion of anantioxidant such as IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™should result in reduced urinary excretion of PGA and increased levelsof glutathione synthesis in blood plasma, thus proving that these threefunctions are interrelated and interdependent.

Thus, analysis of a urine sample for measurement of PGA from a patientexperiencing oxidative stress can be done using standard gaschromatography-mass spectrometry techniques and would demonstrate amarkedly increased excretion of 5-oxoproline or PGA in a patientexperiencing oxidative stress and would correlate with lowered plasmaglutathione levels.

PGA measurements can be done by gas chromatography using aHewlett-Packard 5890 series II fitted with 7673A autosampler, HP-1capillary column (25 m×0.2 mm×0.33-μm film thickness; Hewlett-Packard),and HP5971A mass-selective detector. Helium gas flow rate can be 0.6mL/min (head pressure, 114 kPa). Split injections (ratio, 100:1) can bemade with a 1-μL sample. A one-step temperature program may be run from70 to 296° C. at 7° C./min after an initial time of 0.5 min. The massspectrometer in electron ionization mode, connected directly to thecapillary column outlet, would be operated at 70 eV. Data aquisition canbe carried out in the scan mode from m/z 58 to 550, with dwell time of100 ms. The method of extraction and preparation of urine samples forgas chromatography-mass spectrometry and the method for qualitativelyand quantitatively identifying 5-oxoproline are based on those describedby Tanaka et al. (Tanaka K, West-Dull A, Hine D O, Lynn TB, Lowe T. Gaschromatographic method of analysis for urinary organic acids. I.Retention indices of 155 metabolically important compounds. Clin Chem1980;26:1839-1846; Tanaka K, West-Dull A, Hine D G, Lynn T B, Lowe T.Gas chromatographic method of analysis for urinary organic acids. II.Description of the procedure and its application to diagnosis ofpatients with organic acidurias. Clin Chem 1980; 26:1847-1853). Theresponse factor to the internal standard, isopentanoic acid, could beused to approximate the 5-oxoproline peak as identified by comparisonwith published spectra.

Amino acid measurements, including measurement of glutathione in urine,plasma, and whole-blood hemolysate could be made with a Biotronic LC5001amino acid analyzer (Eppendorf-Netheler-Hinz, division of Biotronic) anda Trivector TRIO computing integrator (Trivector Technical Services). Aglass separation column (3.2×385 mm) could be used with BTC2710 10-μmseparation exchange resin (Eppendorf-Netheler-Hinz) and lithium citrateseparation buffer (flow rate, 0.30 mL/min). Separation temperatureswould be set at 32° C. for 44 min, 34° C. for 28 min, and 60° C. for 31min. For colorimetric peak detection at 570 and 440 nm, 500 μmol/Laminoethyl-L-cysteine hydrochloride in 37 mmol/L lithium/76 mmol/Lcitrate buffer at pH 2.2 would be used as the internal standard. Plasma,urine, and whole-blood hemolysate would be pretreated with crystalline5-sulfosalicylic acid as a deproteinization step.

Treatment Groups

As noted above, it is generally recognized that many disease processesare attributed to the presence of elevated levels of free radicals andreactive oxygen species (ROS) and reactive nitrogen species (RNS), suchas superoxide, hydrogen peroxide, singlet oxygen, peroxynitrite ,hydroxyl radicals, hypochlorous acid (and other hypohalous acids) andnitric oxide. Furthermore, subjects suffering from any of theseconditions may benefit from therapy with anti-oxidants. It is withrespect to these particular diseases and conditions that the currentinvention would be beneficial, particularly with respect to assessingthe need of the patient for treatment with anti-oxidant therapy and formonitoring the effectiveness of such therapy. Moreover, the novel IMMUNEFORMULATIONs described in U.S. Pat. Nos. 6,667,063 and 6,592,908 wouldbe beneficial for treatment of these patients and the amount andduration of therapy with these novel compositions, as well as otheranti-oxidant formulations may be monitored effectively using the methodsof the present invention. A summary of particular diseases for whichanti-oxidant therapy would be beneficial and for which monitoring theneed for and effectiveness of such therapy using the methods describedherein follows.

The methods of the present invention may be utilized to assess anindividual's need for specific therapy with an antioxidant, and as such,may be considered as a means to assess either an individual's currentmedical condition and needs, or alternatively, the methods may be usedas a prophylactic means to allow identification of individuals ofparticularly high risk for diseases known to be associated with highoxidative stress, such as, but not limited to, atherosclerosis andcardiovascular disease. Upon such identification, such subjects canadopt more frequent testing, dietary adjustments, monitoring andregulation of blood pressure, and the like. As a diagnostic assay, themethods of this invention supplement traditional testing methods toidentify subjects known to be at risk who may prove resistant toconventional therapeutic regimens and alter the prescribed treatment.Thus, for example, where a subject is diagnosed with early stages ofatherosclerosis, a positive test using the assays of this invention mayindicate additional drug intervention rather than simply dietary orlifestyle changes.

Cardiovascular Disease

Oxidative modification of low density lipoproteins (LDL) is recognizedas one of the major processes involved in atherogenesis andcardiovascular disease. Thus, a measurement of LDL oxidativesusceptibility could be of clinical significance (Scoccia A E et al, BMCClin Pathol.. (2001) 1(1):1). Furthermore, more precise measurements ofboth lipid oxidation and pyroglutamic acid measurements in the urine, aswell as plasma glutathione measurements as outlined in the presentinvention could be even more beneficial in terms of assessing thepatients' need for treatment with both an anti-oxidant as well as lipidlowering drugs and would also be beneficial in terms of monitoring theutilization efficiency of these drugs as well as the effectiveness oftherapy.

Cancer

The role of oxidative stress in many cancers has been underinvestigation for many years. However, it is recently becoming moreapparent that reactive oxygen or nitrogen species may in fact affectsignaling pathways in many hyperproliferative disorders. For example,prostate cancer (PC) has become the most frequently diagnosed neoplasmand the second leading cause of cancer-related mortality in men. Itsincidence rate has continued to increase rapidly during the past twodecades, especially in men over the age of 50 years as they are livinglonger. The prostate in aging males is highly susceptible to benign andmalignant proliferative changes. It is believed that about two/thirds ofall cancers could have been prevented based upon lifestyle choices. Howenvironment, diet and genetics interact to either induce or preventprostate cancer (PC) is not known. Free radicals play a significant butparadoxical role acting as a “double-edged sword” to regulate cellularprocesses. That is, because of their effect on cell signaling pathwaysand in particular, apoptosis, it appears that ROS may prevent apoptosisand can thus maintain proliferation in certain cancer cells. Thus, thereappears in certain instances to be a paradoxical role for ROS in thesesituations. Recent in vitro studies using benign prostate hyperplasia(BPH) and PC cell lines grown under various oxidative stress conditionsconfirm this theory. Key signal transduction mechanisms may be involvedin ROS induced effects on prostate cell growth, cell-cycle checkpoints,apoptosis and transcription factors. Thus, dietary antioxidants may havea beneficial effect on these mechanisms (Sikka S C Curr Med Chem (2003)10(24):2679-92).

Furthermore, it is also known that many of the therapies available fortreatment of cancers are associated with oxidative tissue damage. Forexample, adriamycin is known to induce cardiac and hepatic toxicity.Studies have been done with specific agents to determine their effectson such peroxidative damage induced by adriamycin (ADR). For example, astudy was conducted to determine the effect of a heparin derivative, lowmolecular weight heparin (LMWH) on the biochemical changes, tissueperoxidative damage and abnormal antioxidant levels in adriamycin (ADR)induced cardiac and hepatic toxicity (Deepa P R et al, Chem Bid Interact(2003) 146(2):201-10). LMWH administration to ADR-induced rats preventedthe rise in serum and tissue levels of LDH, aminotransferases and ALP,while these parameters were significantly elevated in the ADR group incomparison with the control group. Cardiotoxicity indicated by rise inserum CPK in the ADR group was attenuated by LMWH treatment in group IV.LMWH decreased the cardiac and hepatic lipid peroxidation induced byADR. Histologic examination revealed that the ADR-induced deleteriouschanges in the heart and liver tissues were offset by LMWH treatment.Restoration of cellular normalcy accredits LMWH with cytoprotective rolein adriamycin-induced cardiac and hepatic toxicity.

Carboplatin is currently being used as an anticancer drug against humancancers. However, high dose of carboplatin chemotherapy result inototoxicity in cancer patients. Carboplatin-induced ototoxicity isrelated to oxidative stress to the cochlea and inner hair cell loss inanimals. It is likely that initial oxidative injury spreads throughoutthe neuroaxis of the auditory system later. A study was done to evaluatecarboplatin-induced hearing loss and oxidative injury to the centralauditory system (inferior colliculus) of the rat (Husain, K. et al. IntJ Toxicol. (2003) 22(5):335-42). Carboplatin significantly increasednitric oxide and lipid peroxidation, xanthine oxidase, and manganesesuperoxide dismutase activities in the inferior colliculus, but not inthe cerebellum, indicating an enhanced flux of free radicals in thecentral auditory system. Carboplatin significantly depressed the reducedto oxidized glutathione ratio, antioxidant enzyme activities, such ascopper-zinc superoxide dismutase, catalase, glutathione peroxidase,glutathione reductase, and glutathione S-transferase, and enzyme proteinexpressions in the inferior colliculus, but not in the cerebellum, 4days after treatment. The data suggest that carboplatin inducedoxidative injury specifically in the inferior colliculus of the ratleading to hearing loss.

Neurological Diseases and Conditions

Oxidative stress, which is now recognized as accountable for redoxdysregulation involving reactive oxygen species (ROS) and reactivenitrogen species (RNS) plays a pivotal role for the modulation ofcritical cellular functions, notably for cells in the neuronal system(Emerit J, Edeas M, Bricaire F. Biomed Pharmacother. 2004 January;58(1):39-46; Smith J V et al, J. Alzheimer's Dis. (2003) 5(4):287-300;Hayashi T. et al, J Cereb Blood Flow Metab (2003) 23(10):1117-28; Niu KCet al. Clin. Exp. Pharmacol. Physiol. (2003) 30(10):745-51), inparticular, neurons, astrocytes and microglia, such as apoptosis programactivation, and ion transport, calcium mobilization, involved inexcitotoxicity. Excitotoxicity and apoptosis are the two main causes ofneuronal death. The role of mitochondria in apoptosis is crucial.Multiple apoptotic pathways emanate from the mitochondria. Therespiratory chain of mitochondria (oxidative phosphorylation), is thefount of cellular energy, i.e. ATP synthesis, and is responsible formost of ROS and notably the first produced, superoxide anion.Mitochondrial dysfunction (i.e. cell energy impairment, apoptosis andoverproduction of ROS), is a final common pathogenic mechanism in agingand in neurodegenerative disease such as Alzheimer's disease (AD),Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Nitricoxide (NO), an RNS, which can be produced by three isoforms ofNO-synthase in brain, plays a prominent role.

The etiology of neurodegenerative diseases remains enigmatic; however,evidence for defects in energy metabolism, excitotoxicity, and foroxidative damage is increasingly compelling. There is most likely acomplex interplay between these mechanisms. A defect in energymetabolism may lead to neuronal depolarization, activation ofN-methyl-D-aspartate excitatory amino acid receptors, and increases inintracellular calcium, which are buffered by mitochondria. Mitochondriaare the major intracellular source of free radicals, and increasedmitochondrial calcium concentrations enhance free radical generation.Mitochondrial DNA is particularly susceptible to oxidative stress, andthere is evidence of age-dependent damage and deterioration ofrespiratory enzyme activities with normal aging. This may contribute tothe delayed onset and age dependence of neurodegenerative diseases.There is evidence for increased oxidative damage to macromolecules inamyotrophic lateral sclerosis, Huntington's disease, Parkinson'sdisease, and Alzheimer's disease. Potential therapeutic approachesinclude glutamate release inhibitors; excitatory amino acid antagonists,strategies to improve mitochondrial function, free radical scavengers,and trophic factors. All of these approaches appear promising inexperimental studies and are now being applied to human studies. (Beal MF, Ann Neurol. 1995 September; 38(3):357-66)

The role of amyloid beta-peptide (Abeta) in the free-radicaloxidative-stress model of neurotoxicity in Alzheimer's disease (AD) hasreceived much attention recently. Studies have been done to study theeffects of Abeta on intracellular free radical levels. A neuroblastomacell line, which stably expresses an AD-associated double mutation,which exhibits both increased secretion and intracellular accumulationof Abeta when stimulated was utilized in one study. In addition, atransgenic Caenorhabditis elegans constitutively expressing human Abetawas also used. A rise in levels of hydrogen peroxide (H2O2) was observedin both in vitro and in vivo AD-associated transgenic models expressingthe Abeta peptide compared with the wild type controls. Furthermore, anage-dependent increase in H2O2-related ROS was observed in wild type C.elegans, which is accelerated in the AD-associated C. elegans mutant.These results support the hypothesis of the involvement of Abeta and ROSin association with AD (Smith, J. V. and Luo, Y. (2003), J. AlzheimersDis. 5(4):287-3000).

The endoplasmic reticulum (ER), which plays important roles inapoptosis, is susceptible to oxidative stress. Because reactive oxygenspecies (ROS) are robustly produced in the ischemic brain, ER damage byROS may be implicated in ischemic neuronal cell death. A study was donewhereby global brain ischemia was induced in wild-type and copper/zincsuperoxide dismutase (SOD1) transgenic rats and ER stress and neuronaldamage was compared. Phosphorylated forms of eukaryotic initiationfactor 2 alpha (eIF2 alpha) and RNA-dependent protein kinase-like EReIF2 alpha kinase (PERK), both of which play active roles in apoptosis,were increased in hippocampal CAI neurons after ischemia but to a lesserdegree in the transgenic animals. This finding, together with thefinding that the transgenic animals showed decreased neuronaldegeneration, indicates that oxidative ER damage is involved in ischemicneuronal cell death (Hayashi T. et al. (2003), J. Cereb. Blood FlowMetab. 23(10): 1117-1128).

Inflammatory Diseases

The role of oxidative stress in inflammatory diseases has also beeninvestigated. For example, Ramos et al. determined the level of cellularoxidative stress blood markers and the enzymatic system of antioxidantdefense in patients suffering from juvenile rheumatoid arthritis (JRA)(Ramos V A et al., (2000), J Pediatr (Rio J), 76(2): 125-32). This studyincluded 64 patients. The patients were separated in three subtypesbased on the pattern of onset within the first six months of disease:polyarticular, pauciarticular and systemic. The control group included60 patients (38 of female sex) following clinical control to diseases ofnon inflammatory nature, in the same hospital. The plasmatic levels ofmalondialdehyde (MDA), lipoperoxide (LPO), hydroperoxide (HPX),carbonile groups (CG) of proteins and gluthathione and the enzymaticactivities of Superoxide dismutase (SOD), gluthathione peroxidase(GSH-Px) and gluthathione reductase were determined. The results showedthat the group of patients with JRA presented high concentrations oflipid peroxidation products, evaluated by determining the plasmaticlevels of MDA, LPO, and HPX; oxidative damage of the circulate protein,determined by CG contents of plasma proteins; elevation of enzymaticactivity of SOD and GSH-Red; decrease of GSH-Px activity and GSH levels.These results demonstrated the presence of molecular damage thatgenerated oxygen free radicals in the JRA patients. The SOD activity andthe changes of gluthathione redox enzymatic cycle confirm the decreaseof ‘ capacity of cellular defense system against the induced toxicity ofoxidative stress in these patients

Drug Induced Oxidation

The generation of free radicals in vivo can be attributed to manythings. For example, many known drugs can increase the production offree radicals in the presence of increased oxygen tensions. These drugsmay include antibiotics that depend on quinoid groups or bound metalsfor activity (nitrofurantoin), antineoplastic agents as bleomycin,anthracyclines (adriamycin, see above) (Fisher, 1988) and methotrexate,which possess pro-oxidant activity (Gressier et al. 1994). In additionradicals derived from penicillamine, phenylbutazone, some fenamic acidsand the aminosalicylate component of sulphasalazine might inactivateprotease and deplete ascorbic acid accelerating lipid peroxidation(Grisham et al. 1992; Halliwel et al. 1992a; Evans et al. 1994).

Radiation Therapy Induced

Radiotherapy may cause tissue injury as a result of free radicalgeneration. Electromagnetic radiation, such as X rays or gamma rays, andparticulate radiation, such as electrons, photons, neutrons, alpha andbeta particles, may generate primary radicals by transferring theirenergy to cellular components including water. These primary radicalscan undergo secondary reactions with dissolved oxygen or with cellularsolutes.

Smoking Induced

The presence of oxidants in tobacco may play a major role in injuringthe respiratory tract. For example, the oxidants in tobacco smokeseverely deplete intracellular antioxidants in lung cells in vivo. Themechanism for doing so is related to oxidant stress. The oxidantmaterials that are present for a time sufficient to cause damage to thealveoli include aldehydes, epoxides, peroxides, and other free radicals.In addition, nitric oxide, peroxyl radicals and carbon centered radicalsare present in the gas phase, while other radicals are present in thetar phase. Examples of radicals in the tar phase include the semiquinonemoieties derived from various quinones and hydroquinones.Micro-haemorrhages are most probably the cause of iron deposition foundin smokers' lung tissue. This form of iron may lead to the formation ofthe lethal hydroxyl radical from hydrogen peroxide. Furthermore, smokershave elevated amounts of neutrophils in the lower respiratory tract.These may contribute to a further elevation of the concentration of freeradicals.

Providing a Biological Sample for Use in the Methods of the PresentInvention

In particular embodiments the assays are performed using a biologicalsample from the organism/subject of interest. While the assays are ofgreat use in humans, they are not so limited. It is believed similaroxidative damage exists essentially in all mammals and thus the assaysof this invention are contemplated for veterinary applications as well.Thus, suitable subjects include, but are not limited to humans,non-human primates, canines, equines, felines, porcines, ungulates,lagomorphs, and the like.

A suitable biological sample includes a sample of a biological material,which may be selected from a blood sample or urine. As used herein ablood sample includes a sample of whole blood or a blood fraction (e.g.serum or plasma). The sample may be fresh blood or stored blood (e.g. ina blood bank) or blood fractions. The sample may be a blood sampleexpressly obtained for the assays of this invention or a blood sampleobtained for another purpose, which can be subsampled for the assays ofthis invention. In a preferred embodiment, the bodily sample ispreferably plasma or urine.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution, heparinized, concentrated if desired, or fractionatedby any number of methods including but not limited toultracentrifugation, fractionation by fast performance liquidchromatography (FPLC), or precipitation of apolipoprotein B containingproteins with dextran sulfate or other methods. Any of a number ofstandard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris, or the like, at physiological pH canbe used.

Lipid and Water-Soluble Antioxidants

As described below, there are many known lipid and water solubleantioxidants known to have beneficial effects in treatment of variousdisease conditions, whereby high levels of oxidative stress may beresponsible in part for progression of the tissue damage associated withsuch diseases and conditions. It would be beneficial to utilize themethods of the present invention to monitor utilization efficiency andeffectiveness of these therapies should it be determined that a patientwould benefit from such therapies. While these antioxidants are believedto be useful in the treatment of the diseases and conditions describedherein, it is believed that therapy with glutathione precursors, IMMUNEFORMULATION 100™ or IMMUNE FORMULATION 200™ would be the most preferredembodiments.

Lipid Soluble Anti-Oxidants

Lutein: A very active lipid-soluble carotenoid antioxidant (2.3 timeshigher than vitamin E), which is readily absorbed into the serum. Luteinand zeaxanthin are major factors in the prevention of maculardegeneration, which is. the leading cause of blindness in the elderlyand represents 10% of all blindness in humans.

Zeaxanthin: A very active lipid-soluble carotenoid antioxidant (2.8times higher than vitamin E) which is readily absorbed into the serum.Lutein and zeaxanthin are implicated in the prevention of maculardegeneration, which is the leading cause of blindness in the elderly andrepresents 10% of all blindness in humans.

Beta-Cryptoxanthin:—Probably the most active of the lipid solubleantioxidants (3.1 times higher than vitamin E) which is readily absorbedinto the serum.

Lycopene: One of the most active lipid-soluble antioxidants (2.8 timeshigher than vitamin E). Research has indicated that lycopene may be veryimportant in the prevention of prostate cancer.

Alpha-Carotene: A known antioxidant and precursor to vitamin A.Experimental evidence shows that alpha carotene is a strongerantioxidant and cellular differentiating agent than beta carotene andtherefore may be better in preventing cancer.

Beta Carotene: A known antioxidant and precursor to vitamin A, which hasbeen most widely researched and used extensively as a diet supplement.It is a strong cellular differentiating agent, and therefore may preventcancer.

Retinol [Vitamin A]: A known antioxidant and cellular differentiatingagent and Therefore may prevent cancer and many aspects of aging.

Retinyl Palmitate: The retinol ester that is most commonly used indietary

Supplements and Foods as a Source of Vitamin A.

Carotenoid classes: This grouping of carotenoids contain manyuncharacterized carotenoids that most likely are beneficial to health.This value provides a good overall value of the amounts of fruits andvegetables being consumed.

Alpha-Tocopherol (Vitamin E):—One of the best characterized and dietsupplemented lipid-soluble antioxidants. Apart from its antioxidantcapabilities, it has cellular differentiation properties which arebelieved to be good in preventing cancer.

Delta-Tocopherol (Vitamin E): Not much is known about the beneficialeffects of delta-tocopherol to humans, though it is normally found atlower amounts in foods and human serum.

Gamma-Tocopherol (Vitamin E): The major type of vitamin E found in theheart and therefore may be selected for the body because of its uniqueproperties either as an antioxidant or as a differentiation agent.

Ubiquinol [Coenzyme Q10]: Is normally synthesized in cells as part ofthe mitochondrial oxidative phosphorylation system and is present inlipid biomembranes. COQ10 can also be absorbed through the diet and canact as a very active antioxidant and protecting LDL from becomingoxidized.

Water-Soluble Antioxidants

Vitamin C [Ascorbate] (in serum and saliva) Ascorbic acid can directlyscavenge oxidative species as well as generate other oxidizedantioxidants such as vitamin E. However, under conditions where thereare free prooxidant metals around, such as iron and copper, vitamin C'sstrong reductive capacity will catalyze the production of oxidative freeradicals.

Thiols (in serum and saliva) are very active antioxidants and reducingagents. Most serum thiols are found in albumin as indicated by freecysteine and glutathione. Albumin thiols are thought to act assacrificial antioxidants that have little biological consequences ofbeing damaged. Because of their high antioxidant reactivity and highconcentration, albumin thiols act as a major defense against freeradical damage to cell membranes.

Uric acid (in serum and saliva) Uric acid is a methylxanthine (likecaffeine) which stimulates brain activity. It is also known to directlyscavenge oxidative species and chelate prooxidant metals.

Direct and Total Bilirubin: This is considered as a waste product ofheme metabolism. Bilirubin is known to be a very active lipid andaqueous soluble serum antioxidant. Direct (conjugated) bilirubin is theform of bilirubin that can be absorbed and removed from the body in thebile.

IMMUNE FORMULATION 100™ and IMMUNE FORMULATION 200™

The methods described herein contemplate the use of several anti-oxidanttherapies effective at treating various diseases or conditions in whichoxidative stress plays a role. However, a preferred embodiment providesfor the use of IMMUNE FORMULATION 100™ or IMMUNE FORMULATION 200™ foradministration to subjects experiencing oxidative stress or immunedysfunction. These formulations were developed for ,use as a nutritionalsupplement that is beneficial if taken on a daily basis. Theseformulations have the benefit of supplying the glutathione precursorsneeded for resynthesis of glutathione when the levels are depleted dueto disease or metabolic dysfunction. In addition, they are safe and costeffective for those patients in need of daily consumption. Furthermore,they can be formulated as chewable tablets or as nutritive bars orwafers. In another embodiment, it is envisioned that IMMUNE FORMULATION100™ or IMMUNE FORMULATION 200™ can be used in combination with any ofthe anti-oxidants listed in paragraphs [0139] through [0156] of thepresent invention.

IMMUNE FORMULATION 100™

The essential components in IMMUNE FORMULATION 100™ are a selected wheyproduct, colostrum and a non-toxic catalytic quantity of elementalselenium or a water soluble precursor of elemental selenium in an amountsufficient to aid in the production of glutathione. Selenium precursorsare much preferred since they are easier to handle.

Selenium is one of numerous trace metals found in many foods. Seleniummay be employed as one of several non-toxic, water soluble, organic orinorganic selenium compounds capable of being absorbed by the body. Thepresently preferred inorganic selenium compounds are aliphatic metalsalts containing selenium in the form of selenite or selenate anions.However, organic selenium compounds are more preferred because they arenormally less toxic than inorganic compounds. Other selenium compoundswhich may be mentioned by way of examples include selenium cystine,selenium methionine mono- and di-seleno carboxylic acids with aboutseven to eleven carbon atoms in the chain. Seleno amino acid chelatesare also useful. Selenium compounds are utilized in this composition inamounts to provide selected quantities of elemental selenium.

A second component of IMMUNE FORMULATION 100™ is whey. Whey is thecurd-free portion of milk that remains after the production of cheese.“Whey” is a term referring to the serum or watery part of milk afterremoval of the cheese. Removal of a substantial portion of the waterresults in a dry whey. There are two common types of dry whey. These aredry whey concentrates and dry whey isolates. The former (WPC) is anoff-white to cream colored product which, depending on the method ofmanufacture, may contain from about 15% to 85% protein based on thetotal weight It may additionally contain small amounts of minerals,vitamins and carbohydrates. Whey protein isolate (WPI) contains morethan 85% by weight of protein. Both types of whey are available fromProliant, Manhattan, Ill.; Davisco Foods International, Inc. EdenPrairie. Minn. or Land-O-Lakes Tulare, Calif. The whey product may be upto about 35% denatured. The whey product may be completely denatured,but the cost of wholly denatured whey is such that it is not feasible toemploy wholly undenatured whey in compositions to serve general humanconsumption or in animal needs. Accordingly, the dried whey productutilized in IMMUNE FORMULATION 100™ will be a whey product concentrateor whey product isolate which is up to about 35% denatured or,conversely about 65% to about 100% undenatured. Preferably, it willcontain from about 65% to about 85% protein. It may comprise from about5% to about 95% of the composition based on the total weight of thecomposition.

A third component of IMMUNE FORMULATION 100™ is colostrum. Colostrum isa thin milky fluid secreted by the mammary gland of mammals a few daysbefore or after parturition. It is a unique combination of beneficialnutrients including protective antibodies, fat, carbohydrate, vitaminsand minerals. The immunological components of colostrum include IgG, IgMand IgA. These components confer passive immunity to the neonate andprotection against infection during the initial period afterparturition. After this period, colostrum is no longer absorbed throughthe gut and the newborn must depend upon its own developing immunesystem for protection. Colostrum is an important factor in the growth ofmammals including humans, bovines, caprines, porcines and equines. Thepreferred colostrums for use in the compositions of this invention arebovine and caprine. Several colostrum products useful in thisformulation are commercially available.

The daily effective dosage of the products of this invention will dependupon the size of the individual (human or animal) being treated, thecondition being treated, the age of the individual and other factorswell known to the physician or veterinarian in. attendance. The optimumdaily dosage can easily be determined by a few simple observations. Itwill generally vary from about 250 mg to 2000 mg per day for humans andsmall animals. For large animals the daily dosage will normally be fromabout 500 mg to 5000 mg per day. While these are projected dose ranges,the methods of the present invention can be utilized to measureutilization efficacy and ultimate effectiveness of these formulations.

IMMUNE FORMULATION 200™

The essential components of IMMUNE FORMULATION 200™ are precursors ofglutathione, namely glutamic acid, cystine or another cysteine precursorand glycine, together with a catalytic quantity of a selenium source.The separate components serve as precursors with selenium for themetabolic formation of glutathione after they have been transportedacross the mucous membrane. The glutathione precursors in thisformulation, which are a mixture of glutamic acid, cystine or anotherrelated cystine precursor, and glycine are in a molar ratio of about1:0.5:1, the amount of glutathione precursors being effective toincrease the content of glutathione in the body tissue of the mammalabove that of a pretreatment level thereby to enhance immune activity.This material is further described and claimed in U.S. Pat. No.6,592,908. The composition may be used alone, but normally it will beemployed in association with one or more non-toxic pharmaceuticallyacceptable carriers appropriate to the method of administration. If anexcess of any amino acid is used, it will presumably be of nutritionalvalue or may simply be metabolized.

IMMUNE FORMULATION 200™ will be utilized to increase the formation ofglutathione and thus to enhance the immune activity of a mammal in needof such treatment. The effect of the treatment is such that after thetreatment, the mammal will be more resistant to microbial infection orother trauma, diseases, or conditions adversely affecting immuneactivity than before such treatment.

Because of its ability to increase production of glutathione, IMMUNEFORMULATION 200™ is useful to treat a wide variety of diseases orconditions associated with the presence of excess free radical orreactive oxygen or nitrogen species. These include, for example, cancer,Alzheimer's disease, arteriosclerosis, rheumatoid arthritis and otherautoimmune diseases, cachexia, coronary artery disease, chronic fatiguesyndrome, AIDS and others as described herein.

The components of this composition are amphoteric and therefore may beemployed as non-toxic metal salts or acid addition salts. Typically, thesalts are alkalic or alkaline earth metal salts, preferably sodium,potassium or calcium salts. Suitable acid addition salts include saltsof hydrochloric, phosphoric and citric acid. The amino acids may also beemployed in the form of certain of their derivatives including estersand anhydrides which before or after transport through the mucousmembrane will be modified into the form in which they will be joinedtogether to form glutathione. All amino acids employed, except glycinewhich does not form optical isomers, are in the natural or L-form.Although wide variations are possible, it will be apparent that theoptimum ratio of glutamic acid to cystine to glycine in this novelcomposition described herein is 1:0.5: 1. If an excess of any acid isused, it will presumably be of nutritional value or may simply bemetabolized.

It is important for the use of this composition that the selenium asemployed in the composition be capable of transport through the mucosalmembrane of the patient under treatment. For this reason, waterinsoluble selenium compounds are not generally useful.

For convenience, the term “selenium” is sometimes used hereinafter toinclude any of the various water soluble selenium products which can betransported through the . mucosal membrane in the practice of thisinvention. It will be understood, however, that the particular forms ofselenium compounds set forth herein are not to be considered limitative.Other selenium compounds, which exhibit the desired activity and arecompatible with the other components in the mixture and are non-toxic,can be used in the practice of the invention. Many of them are availablecommercially.

In fact, the amount of selenium precursor employed in this novelcomposition is only enough to provide a catalytic quantity of theelement to activate the glutathione system. The catalytic quantity ofselenium precursor utilized in the compositions of this invention issuch that it will produce either in one dosage unit or in multipledosage units sufficient elemental selenium to promote the production andactivation of glutathione.

Typically, this will be at or near the recommended daily allowance ofselenium for the individual mammal under treatment. This amount will bewell below the toxicity limit for elemental selenium. By way ofnon-limiting examples, a representative range of catalytic quantities ofselenium precursors is based on the age of the individual. Therecommended daily allowances for elemental selenium as reported in ThePharmacological Basis of Therapeutics, Ninth Edition, page 1540, TheMcGraw-Hill Companies, 1996. The recommended daily dosage for humanstherefore ranges from 10 to 75 μg per day. For animals the range maydepend upon the animal and its size.

The tablets or wafers, with fillers will typically weigh from about 0.5to 5 grams and will contain a therapeutically effective amount of theessential ingredients together with the selected vehicle. Tablets andother forms of the immunoenhancing compositions can be prepared toprovide any quantity of elemental selenium from less than 1 μg to 7.5μg. For example, a tablet containing 10 μg of selenium methionine iscapable of delivering 4 μg of elemental selenium, and 7.5 μg of seleniummethionine is capable of delivering 3 μg of selenium. :Tablets may begiven several times per day to achieve .the desired immune enhancingeffect.

A one a day tablet weighing two grams may contain 200 mg or more of thecomposition. A similar tablet intended to be used every four hours maycontain 50 mg to 100 mg or more of the therapeutically effectivecomposition.

Immune Function Analysis

The methods of the present invention provide for measurement of specificimmune cell numbers and activities, including quantitation of specific Tcell subsets and function of natural killer cells. In particular, CD4+ Tcells and CD8+ T cells provide immune protection from all forms ofpathogens, including bacteria, viruses, and tumors. These cells act as asource of cytokines/lymphokines for induction of cytolytic T cells, oneof the primary immune cells that lyse virus infected target cells aswell as tumor targets. These cells also secrete factors that aid in theinduction of specific B cell or antibody producing cell populations.

Assays for measuring the numbers of these cell populations are known tothose skilled in the art. For example, as shown below, the most commonlyused procedures are by FACS analysis, whereby the cell populations areincubated with labeled antibodies specific for cell surface markers.These antibodies may be labeled with phycoerythrin or FITC and after aperiod of time, they are washed and analyzed in a fluorescent activatedcell sorter. Alternatively, assays can be set up to measure the activityof these cells in a specific chromium release assay to assess theiractivity. These assays are also known to one skilled in the art.

Natural killer (NK) cells are one of the early defense mechanisms in thebody for protection against a variety of pathogens. These cells may alsobe assessed by the use of specific cell surface markers and FACSanalysis. Alternatively, their activity may be assessed using NKsensitive target cells in a chromium release assay as described below.

Assay Formats

The methods of this invention may use assays which may be practiced inalmost a limitless variety of formats depending on the particular needsat hand. Such formats include, but are not limited to traditional “wetchemistry” (e.g. as might be performed in a research laboratory),high-throughput assay formats (e.g. as might be performed in a pathologyor other clinical laboratory), and “test strip” formats, (e.g. as mightbe performed at home or in a doctor's office).

Traditional Wet Chemistry

The assays of this invention can be performed using traditional “wetchemistry” approaches. Basically this involves performing the assays asthey would be performed in a research laboratory. Typically the assaysare run in a fluid phase (e.g. in a buffer with appropriate reagents(e.g. lipids, oxidized lipids, oxidizing agent, etc.) added to thereaction mixture as necessary. The oxidized lipid concentrations areassayed using standard procedures and instruments, e.g. as described inthe examples.

High-throughput Assay Formats

Where population studies are being performed, and/or inclinical/commercial laboratories where tens, hundreds or even thousandsof samples are being processed (sometimes in a single day) it is oftenpreferably to perform the assays using high-throughput formats. Highthroughput assay modalities are highly instrumented assays that minimizehuman intervention in sample processing, running of the assay, acquiringassay data, and (often) analyzing results. In particular embodiments,high throughput systems are designed as continuous “flow-through”systems, and/or as highly parallel systems.

Flow through systems typically provide a continuous fluid path withvarious reagents/operations localized at different locations along thepath. Thus, for example a blood sample may be applied to a samplereceiving area where it is mixed with a buffer, the path may then leadto a cell sorter that removes large particulate matter (e.g. cells), theresulting fluid may then flow past various reagents (e.g. where thereagents are added at “input stations” or are simply affixed to the wallof the channel through which the fluid flows. Thus, for example, thesample may be sequentially combined with a lipid (e.g. provided as anLDL), then an oxidation agent, an agent for detecting oxidation, and adetector where a signal (e.g. a calorimetric or fluorescent signal) isread providing a measurement of oxidized lipid.

In highly parallel high throughput systems samples are typicallyprocessed in microtiter plate formats (e.g. 96 well plates, 1536 wellplates, etc.) with computer-controlled robotics regulating sampleprocessing reagent handling and data acquisition. In such assays, thevarious reagents may all be provided in solution. Alternatively some orall of the reagents (e.g. oxidized lipids, indicators, oxidizing agents,etc.) may be provided affixed to the walls of the microtiter plates.

In a particular embodiment of the present invention, it is envisionedthat all three products, that is, lipid peroxides, pyroglutamic acid andglutathione may be measured concurrently. For example, a 96 well platemay be prepared that contains wells to which antibodies have beenattached for each of the three products. Thus, one set of thirty twowells would contain an antibody to lipid peroxides, one set of wellswould contain an antibody to glutathione, and a third set of thirty twowells would contain an antibody prepared to pyroglutamic acid. Thus,urine samples can be applied to the wells containing the antibodies tolipid peroxides and to those wells containing antibodies to pyroglutamicacid. Whole blood or blood plasma can be added to those wells containingantibodies to glutathione. After an appropriate incubation time, forexample, 1 hour at 37° C., the plates would be washed, and secondaryantibodies which are conjugated to (labeled with) an enzyme orfluorophore can be added, incubated in the same manner, and the amountof secondary label bound can be measured using a substrate for theenzyme or if the fluorophore method is used, the amount of label boundis measured using spectrophotometric techniques at the appropriatewavelength for the fluorophore.

High throughput screening systems that can be readily adapted to theassays of this invention are commercially available (see, e.g., ZymarkCorp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass., etc.). These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols. Thus, for example, Zymark Corp. provides technical bulletinsdescribing screening systems for detecting the modulation of genetranscription, ligand binding, and the like.

“Test Strip” Assay Formats

The methods of the present invention may also utilize assays which areprovided in “test well” or “test strip” formats. In “test well” or “teststrip” formats, the biological sample is typically placed in the well orapplied to a receiving zone on the strip and then a fluorescent orcalorimetric indicator appears which, in this case, provides a measureof the protection or repair afforded by the subject's HDL or componentsthereof.

Many patents have been issued which describe the various physicalarrangements for blood testing. These include systems which involvelateral or horizontal movement of the blood, as well as plasma testing.For example, U.S. Pat. Nos. 4,876,067, 4,861,712, 4,839,297, and4,786,603 describe test carriers and methods for analyticaldetermination of components of bodily fluids, including separatingplasma from blood using glass fibers and the like. These patents, allteach systems which require some type of rotation of test pads or aportion of the test pads during use. U.S. Pat. No. 4,816,224 describes adevice for separating plasma or serum from whole blood and analyzing theserum using a glass fiber layer having specific dimensions andabsorption to separate out the plasma from the whole blood forsubsequent reaction. Similarly, U.S. Pat. No. 4,857,453 describes adevice for performing an assay using capillary action and a test stripcontaining sealed liquid reagents including visible indicators. U.S.Pat. No. 4,906,439 describes a diagnostic device for efficiently andaccurately analyzing a sample of bodily fluid using fluid delivery in alateral movement via flow through channels or grooves.

Kits

The methods of the present invention provide for measuring the amountsof specific markers of oxidative stress. In a particular embodiment, atleast three markers are quantitated using standard reagents and kits,which are commercially available to measure each marker individually. Inanother particular embodiment, the methods further comprise themeasurement of the number and/or activity of specific immune cellpopulations, preferably T cells and natural killer cells usingtechniques known to those skilled in the art. Thus, the presentinvention provides a more quantitative and accurate means of assessing asubject's need for antioxidative therapy by measuring all of theseparameters concurrently. To the inventor's knowledge, no other artcurrently exists which describes combining the concurrent non-invasivetechniques and measurements described herein for assessing the need for,and to measure the effectiveness of, therapy with anti-oxidants.

However, one of the oxidative markers, pyroglutamic acid, is measuredusing non-immunological techniques, in particular, gas chromatographyand mass spectrometry are used. While this procedure provides theaccuracy and sensitivity that is needed for such measurements in thepresent invention, the procedure can be time consuming and requires theuse of very specialized equipment. Accordingly, the present inventionalso provides for kits comprising binding partners for the oxidativemarkers and the reagents needed for detection of the oxidative stressmarkers. The kits of the present invention provide advantages over thosecommercially available in that at least three oxidative markers can bemeasured concurrently using the same assay format. In anotherembodiment, the kits may contain binding partners for the threeoxidative stress markers, that is, for lipid peroxide, pyroglutamic acidand glutathione, as well as binding partners for cell surface markers onCD4+ T cells, CD8+ T cells and a cell surface marker for natural killercells, such as NK1.1/CD69. Thus, a kit of the present invention may beuseful for monitoring both markers of oxidative stress as well asmarkers for immune cells known to be beneficial against known pathogens.

Thus, an assay format is preferred in which binding partners such asantibodies can be obtained or prepared for the analytes (lipid peroxide,pyroglutamic acid, glutathione, CD4, CD8, NK1.1/CD69). Biotin-avidin,biotin-streptavidin or other biotin-binding-reagent reactions can beused to enhance or modulate the test. However, any such assay can bedevised using other binding partners to the analytes (oxidative stressmarkers and immune cell markers), including but not limited toextracellular or intracellular receptor proteins which recognize theanalytes, binding fragments thereof, hybridization probes for nucleicacids, lectins for carbohydrates, etc. The particular selection ofbinding partners is not limiting, provided that the binding partnerspermit the test to operate as described herein. The preselectedanalytes, when present, are detectable by binding two binding partners,one immobilized on the test strip (or whatever format the assay isprovided) and another part of a conjugate. This is taken intoconsideration in the selection of the reagents for the assay.

The dry test strip may be set up in any format in which contact of thesample with the reagents is permitted and the formation and mobility ofthe immunocomplexes and other complexes forming therein are permitted toflow and contact an immobilized reagent at the capture line. Variousformats are available to achieve this purpose, which may be selected bythe skilled artisan.

The label portion of the mobile, labeled antibody to the marker may be avisible label, such as gold or latex, an ultraviolet absorptive marker,fluorescent marker, radionuclide or radioisotope-containing marker, anenzymatic marker, or any other detectable label. A visibly detectablemarker or one that can be easily read in a reflectometer is preferred,for use by eye, reading or confirmation with a reflectometer. Otherlabels may be applicable to other semi-automated or automatedinstrumentation.

The conjugates of the invention may be prepared by conventional methods,such as by activation of an active moiety, use of homobifunctional orheterobifunctional cross-linking reagents, carbodiimides, and othersknown in the art. Preparation of, for example, a gold-labeled antibody,a conjugate between an antibody and an analyte (not an immunocomplex buta covalent attachment which allows each member to independently exhibitits binding properties), biotinylation of an antibody, conjugation ofstreptavidin with a protein, immobilization of antibodies on membranesurfaces, etc., are all methods known to one of skill in the art.

A kit may have at least one reagent for carrying out an assay of theinvention, such as a kit comprising a conjugate between a biotin-bindingreagent and an antibody to an oxidative marker. Preferably, the kitcomprises all of the reagents needed to carry out any one of theaforementioned assays, whether it be homogeneous, heterogeneous,comprise a single conjugate of the marker conjugated to an antibody tothe analyte, or comprise two reagents which serve this function (such asa biotinylated antibody to the analyte plus a streptavidin-markerconjugate, or a biotinylated marker plus a streptavidin conjugated to anantibody to the analyte conjugate), or whether the assay employs animmobilized antibody to the analyte and a labeled antibody to adifferent site on the analyte. Referring to the first analyte as analyteand the second analyte as marker, and a second binding partner as abinding partner which recognizes a different epitope than the firstbinding partner mentioned, the following kits are non-limiting examplesof those embraced herein:

In the foregoing kits, the binding partners are preferably antibodies orbinding portions thereof, and both the binding partner to the anal ytes(the three oxidative stress markers, and the markers for immune cells)and the second binding partner to the analytes capable of simultaneouslybinding to the analyte. The immobilized binding partner may be providedin the form of a capture line on a test strip, or it may be in the formof a microplate well surface or plastic bead, by way of non-limitingexamples. The kits may be used in a homogeneous format, wherein allreagents are added to the sample simultaneously and no washing step isrequired for a readout, or the kits may be used in a multi-stepprocedure where successive additions or steps are carried out, with theimmobilized reagent added last, with an optional washing step.

The antibodies specific for the three oxidative stress markers may beobtained commercially, or can be produced by techniques known to thoseskilled in the art.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how touse the methods described herein, and are not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Assessment of the Effects of IMMUNE FORMULATION 100™ or 200™on Adriamycin Induced Oxidative Stress Levels in Rats

Adriamycin, while being an effective anti-cancer agent in humans, isknown to induce nephropathy in a rat model (Zima, T. et al (1998),Nephrol. Dial. Transplant, 13: 1975-1979). Its deleterious effects onthe kidney are believed to be due in part on the generation of reactiveoxygen species (ROS) following its metabolism in vivo. Thus, theadministration of adriamycin in a rat model provides an opportunity tostudy the effects of this compound on the generation of reactive oxygenspecies by measuring the by-products of such ROS, such as urinary lipidperoxides and pyroglutamic acid, as well as plasma glutathione.Furthermore, one can also use this model to look at the effects of thistoxic compound on kidney damage, and on numbers of immune cellpopulations and on natural killer cell activity. In addition, this modelprovides an opportunity to assess the need for treatment of an animalwith an anti-oxidant, as well as monitoring the utilization efficiencyof such treatment, and ultimately, to monitor the effectiveness of thistreatment. Accordingly, this model will be used to study the effects ofIMMUNE FORMULATION 100™ or 200™ on the normalization of urinary lipidperoxides, pyroglutamic acid and on plasma glutathione. Studies willalso be done to measure the effects on CD4+ and CD8+ T cell populations,as well as on natural killer cell activity.

Materials and Methods Rats

Ninety female Sprague-Dawley rats (200-225 g) are obtained from CharlesRiver and acclimatized for 7 days after delivery. Animals are housed incommunal cages, fed a rat maintenance diet and water ad libitum. Duringexperiments, animals are housed in individual metabolism cages designedto separate and collect feces and urine, and given powdered diet andwater ad libitum. Lighting is controlled to give a regular 12 h light-12h dark cycle; room temperature is maintained at 21±1° C. Urine samples(24 h) are collected over ice and centrifuged (2000 r.p.m., 10 min, 4°C.) to remove hair and food debris and stored (−80° C.) in aliquots forlater analysis. The general condition of the animals is monitored dailyand rats are weighed twice a week.

Study Design

The rats are randomized into three groups of thirty rats per group.Group I is designated the Placebo control group, Group II is designatedthe Adriamycin only group, and Group HI is designated the Adriamycinplus IMMUNE FORMULATION 100™ or 200™ treatment group. Twenty-four hoursprior to dosing, 1.0 ml of whole blood is collected from the tail veinof each rat and placed into a heparinized microcentrifuge tube.Twenty-four hour urines are also collected from each rat prior todosing, and the urines are centrifuged to remove any food or hair andfrozen at −80° C. until assayed for the metabolites lipid peroxide andpyroglutamic acid. Plasma is separated from whole blood bycentrifugation (2500×g for 25 min at 4° C.) and stored at −80° C. forglutathione analysis.

On day 1, the rats in Groups II and III are given Adriamycin at 5 mg/kgi.v., while the rats in Group I are injected with I.V. saline asplacebo. On day 2, the rats in Group III are given either IMMUNEFORMULATION 100™ or 200™. IMMUNE FORMULATION 100™ will be given in adose ranging from 5 to 125 grams per day prepared in powdered rat chow.IMMUNE FORMULATION 200™ will be given in a dose ranging from 25 to 100mg per day by oral gavage. The rats in group HI are dosed daily with theIMMUNE FORMULATIONs at the doses described. On day 4 and every threedays afterwards, 1.0 ml of whole blood is collected from the tail veinof each rat in the study, and 24 hour urines are also collected. Theurines and blood are treated as described above, and frozen away at −80°C. for future analysis of lipid peroxides and pyroglutamic acid.

The urine samples and plasma samples are analyzed on day seven for lipidperoxide, pyroglutamic acid and glutathione, using the methods describedbelow. If the levels of Group III remain outside of the range of theplacebo group, the rats in Group III are dose adjusted in an incrementof 10 gram doses for IMMUNE FORMULATION 100™ and in increments of 10 mgdoses for IMMUNE FORMULATION 200™, and dosing is continued for anotherweek. After the rats in Group III have been dosed for a total of 2weeks, the plasma and urine levels are again assessed for glutathione,lipid peroxide and pyroglutamic acid using the methods described below.If the ranges of all three are still outside of the range of the placebotreated Group I rats, the levels of the IMMUNE FORMULATIONs are againscaled up as described above and dosing continues another week. Afterone month of following this testing and dosing regimen, a final 24 hoururine sample is collected and a final bleed is done prior to sacrificeof the rats by CO2 asphyxiation. Spleens are removed and single cellsuspensions are prepared for use in natural killer cell assays and forstaining with markers specific for CD4+ and CD8+ T cells.

Urinary Lipid Peroxide Measurement by Determination of UrinaryThiobarbituric-acid-reacting Substances (TBARS)

Lipid peroxide levels in urine samples are measured colorimetrically bythe thiobarbituric acid reaction (TBARS) as described in the followingreferences: Buege J A, Aust S D, Methods Enzymol 1978, 52:302-310 and inValenzuela et al (Valenzuela A: Life Sci 1991, 48:301-309). The level oflipid peroxides in urine is expressed as equivalents of malondialdehyde(MDA). Malondialdehyde standards are freshly prepared fromtetraetoxypropane and treated in the same way as the urine samples.Briefly, 200 μl of urine is combined with 10 μl of 5% butylatedhydroxytoluene (in glacial acetic acid) and 300 l of a 0.5% aqueousthiobarbituric acid (TBA) solution. The samples are vortexed and areincubated at 100° C. for 30 minutes, and the absorbance at 532 nm ismeasured using a PerkinElmer Lamba 3B spectrophotometer (PerkinElmer,Wellesley, Mass., USA). The quantity of TBARS is proportionate to theamount of MDA, a lipid peroxidation product generated by the oxidationof membrane lipids by reactive oxygen species. MDA reacts with TBA toform a 1:2 MDA—TBA adduct that absorbs at 532 nm. To control for urineconcentration, data is normalized to urine creatinine concentrations, asdescribed (Coulthard M G, Hey E N, Ruddock V: Early Hum Dev 1985,11:11-19). Creatinine can also be measured by a Sigma diagnostics kit555-A.

Urinary Pyroglutamic Acid Measurement

Using a range of 1 and 2 dimensional 500 MHz 1H NMR spectroscopictechniques, solid phase extraction and mass spectrometry, the metabolitepyroglutamic acid (PGA), also known as 5-oxoproline (5OXP), can bemeasured in the urine. (Ghauri F Y, et al. (1993), Biochem Pharmacol.,September 1; 46(5):953-7.) Alternatively, PGA measurements can be doneby gas chromatography using a Hewlett-Packard 5890 series II fitted with7673A autosampler, HP-1 capillary column (25 m×0.2 mm×0.33-μm filmthickness; Hewlett-Packard), and HP5971A mass-selective detector. Heliumgas flow rate can be 0.6 ml/min (head pressure, 114 kPa). Splitinjections (ratio, 100:1) can be made with a 1-μL sample. A one-steptemperature program may be run from 70 to 290° C. at 7° C./min after aninitial time of 0.5 min. The mass spectrometer in electron ionizationmode, connected directly to the capillary column outlet, would beoperated at 70 eV. Data aquisition can be carried out in the scan modefrom m/z 58 to 550, with dwell time of 100 ms. The method of extractionand preparation of urine samples for gas chromatography-massspectrometry and the method for qualitatively and quantitativelyidentifying 5-oxoproline are based on those described by Tanaka et al.(Tanaka K, West-Dull A, Hine D G, Lynn TB, Lowe T. Gas chromatographicmethod of analysis for urinary organic acids. I. Retention indices of155 metabolically important compounds. Clin Chem 1980; 26:1839-1846;Tanaka K, West-Dull A, Hine D G, Lynn T B, Lowe T., Gas chromatographicmethod of analysis for urinary organic acids. H. Description of theprocedure and its application to diagnosis of patients with organicacidurias. Clin Chem 1980; 26:1847-1853). The response fator to theinternal standard, isopentanoic acid, could be used to.approximate the5-oxoproline peak as identified by comparison with published spectra.

Plasma Glutathione Measurement

A GSH kit can be procured from Calbiochem. Plasma samples are defrostedand serial dilutions prepared and analyzed for total GSH per themanufacturer's instructions (Calbiochem).

Natural Killer Cell Assay

NK function (i.e., activity) can be measured by ⁵¹Cr releasecytotoxicity assays against a suitable target cell. An example of asuitable target cell by which to measure NK cell cytotoxic activity isYAC-1. NK cell activation can also be measured by determining anupregulation of NK1.1/CD69 on cells in various organs, including spleen,lymph node, lung and liver, by flow cytometric analysis .

Cytotoxicity Assay

A standard 4-hour ⁵¹Cr-release assay is used to quantitate cytotoxicactivity. present in freshly isolated spleen mononuclear cells, usingYAC-1 cells as targets. Briefly, effector cells from spleen are added indecreasing concentrations to duplicate wells of a Linbro plate, to whichwas then added 5×10³ target cells that had been previously labeled for 1hour with ⁵¹Cr. The plates are incubated at 37° C. for 4 hours, thensupernatants from each well are harvested and the amount of radioactive⁵¹Cr present is determined by automated gamma counter. For spontaneousrelease, only targets were added and the well was made up to theequivalent volume with medium, for maximum release 0.1 ml of 2% SDS wasadded to wells containing targets only. The percentage specific lysis iscalculated as ((experimental release-spontaneous release)/(maximumrelease-spontaneous release))×100.

Flow Cytometry

Upregulation of the early activation marker, CD69, which is upregulatedon activated T cells, B cells, macrophages and NK cells, can be used toassess early immune cell activation. Single cell suspensions areprepared from spleens of rats by NH₄Cl lysis procedure (Sambrook,supra). Cells are analyzed using a Becton-Dickinson FACSCalibur flowcytometer (Becton Dickinson, Mountain View, Calif.), with analysis gatesset by first gating on spleen lymphocytes. Between 10,000 and 30,000gated events are analyzed for each cell type. For analysis of cellactivation, 3-color flow cytometric analysis may be done, usinganti-CD69 phycoerythrin (Pharmingen, San Diego, Calif.) to quantitatethe number of CD69 positive cells. Cells can also be dual-labeled toevaluate T cells (anti-αβTCR antibody (biotin H57.597; Pharmingen) plusantibodies to either CD4 (FITC RM4-5; Pharmingen) or CD8 (FITC 53-6.7;Pharmingen). NK cells can be dual-labeled using anti NK 1.1 (biotinPK136; Pharmingen) and anti CD3 (FITC 2C11). The percentage of doublepositive cells expressing CD69 can be determined for each cell type, andthe mean (+/−SD) CD69+ cells plotted.

Assessment of CD4+ and CD8+ T Cell Numbers

Monoclonal antibodies (mAbs) to murine CD4, GK1.5 (ATCC TIB 207) andmurine CD8, 53-6.72 (ATCC TIB 105) are purified from hybridoma culturesupernatants over a recombinant protein G column (Pharmacia, Piscataway,N.J.). As a control, purified rat IgG is purchased from Calbiochem (SanDiego, Calif.). Flow cytometry is performed as described above to assessthe effectiveness of the treatment regimen. Standard flow cytometrictechniques are used using Phycoerythrin labeled anti-rat CD4 and FITClabeled anti-rat CD8 purchased, for example, from Pharmingen (San Diego,Calif.).

Statistical Analyses

Significant differences between groups can be determined by theTukey-Kramer HSD multiple comparisons test using JMP® statisticaldiscovery software (SAS Institute Inc., Cary, N.C, or by an Analysis ofVariance (ANOVA) with a Bonferroni P value for multiple comparisons.

Example 2 IMMUNE FORMULATION 100™ Tablet Formulation

Ingredients: Whey (PROLIANT ™ 8010 or 8200) 1 gm Colostrum 1 gm Seleniummethionine 5 μg

Blend the ingredients together and pass through a 60 mesh screen andtumble until the components are thoroughly mixed. Compress using a 7/16inch standard concave punch.

Example 3 IMMUNE FORMULATION 100™ Powder Formulation

Ingredients: Whey (Proliant ™ 8010 or 8200) 75 gm Colostrum 25 mgSelenium methionine 15 μg

Thoroughly mix the ingredients in a blender and pass through a 80 meshscreen.

This powder may be used for mixing with animal feeds, frostings, fruitspreads and beverages to be pasteurized.

Example 4 IMMUNE FORMULATION 100™ Chewable Tablet Formulation

Ingredients: Vitamin A USP (dry, stabilized form) 500 USP units VitaminD (dry, stabilized form) 400 USP units Ascorbic Acid USP 60.0 mg Thiamine Hydrochloride USP   1 mg Riboflavin USP 1.5 mg PyridoxineHydrochloride USP   1 mg Cyanocobalamin USP 2 μg Calcium PantothenateUSP   3 mg Niacinamide USP  10 mg Mannitol USP (granular) 236.2 mg  CornStarch 16.6 mg  Sodium saccharin 1.1 mg Magnesium stearate 6.6 mg TalcUSP 10 mg Whey (Proliant ™ 8010) 8 g Colostrum  500 mg  Seleniummethionine 7 μg

Thoroughly mix the ingredients in a blender and compress using a 3/8inch bevel-edge punch.

Example 5 Immune Formulation 200™ (Tablet)

Ingredients:  89 mg cystine  75 mg glycine 147 mg glutamic acid 22.5 μgpolyvinylpyrolidone 61.25 mg   lactose 4.5 ml alcohol SD3A-200 proof  9mg stearic acid 42.3 mg  corn starch   10 μg selenium methionine

Blend the cystine, glycine, glutamic acid, polyvinylpyrrolidone andlactose together and pass through a 40 mesh screen. Add the alcoholslowly and knead well. Screen the wet mesh through a 4 mesh screen. Drythe granulation at 50 degrees centigrade for 10 hours. Pass the mixtureof stearic acid, corn starch and selenium compound through a 60 meshscreen and tumble with the granulation until all the ingredients arewell mixed. Compress using a 7/16 inch standard concave punch.

Example 6 Immune Formulation 200™ (Tablet)

Ingredients: 178 mg cystine 150 mg glycine 294 mg glutamic acid 5 μgselenium methionine 126 mg lactose  78 mg potato starch  96 mg ethylcellulose  54 mg stearic acid

Thoroughly mix the ingredients in a blender, dry, put through a 12 meshscreen and compress into tablet using a 13/32 inch concave punch.

We claim:
 1. A method for assessing the need for treatment of a subjectwith an anti-oxidant comprising the steps of: a) collecting a sample ofbody fluid from a subject suspected of needing suchtreatment; b)measuring the amount of lipid peroxide and pyroglutamic acid levels insaid sample; c) measuring the level of blood plasma glutathione; d)comparing the amount of lipid peroxide and pyroglutamic acid in saidsample with that of a normal standard; and e) comparing the level ofblood plasma glutathione with that of a normal standard; and wherein thepresence of lipid peroxide and pyroglutamic acid in said sample and theblood plasma levels of glutathione are present in amounts that lieoutside the normal range are indicative of a need for anti-oxidanttreatment.
 2. The method of claim 1, wherein said subject in need oftreatment with an anti-oxidant also experiences a reduction in immunecell number and/or function.
 3. The method of claim 2, wherein saidimmune cell is selected from the group consisting of a T cell, a B cellor a natural killer cell.
 4. The method of claim 3, wherein said T cellis selected from the group consisting of a CD4+ T cell or a COB+ T cell.5. The method of claim 1, wherein said anti-oxidant comprises aformulation consisting of a glutathione precursor.
 6. The method ofclaim 1, wherein the sample of body fluid is urine. 7-28. (canceled) 29.A kit for measuring oxidative stress in a subject comprising: a) a solidsubstrate containing immobilized binding partners specific for at leastthree markers for oxidative stress; b) either: i) an enzyme conjugatedsecond binding partner to the oxidative stress markers; or ii) abiotinylated second binding partner to the oxidative stress markers; c)either: i) the enzyme substrate and the developing reagents specific forthe enzyme conjugated second binding partner from step b) i); or ii) astreptavidin conjugated third binding partner specific for the secondbinding partner of step b) ii); d) buffers for washing and sampledilution; e) standards for each of the at least three markers ofoxidative stress; and f) instructions for use of said kit.
 30. The kitof claim 29, further comprising additional binding partners specific forcell surface markers for CD4+ T cells, CD8+ T cells and natural killercells.
 31. The kit of claim 29, wherein said markers of oxidative stressare selected from the group consisting of lipid peroxide, pyroglutamicacid and glutathione.
 32. The kit of claim 29, wherein said bindingpartner is an antibody selected from the group consisting of amonoclonal antibody, a polyclonal antibody, a chimeric antibody, and anycombination thereof. 33-36. (canceled)
 37. The method of claim 1,comprising determining the amount of lipid peroxides and pyroglutamicacid in the sample with the Wheeler method, iron thiocyanate method,thiobarbituric acid method or with a combination of a peroxidase and ahydrogen donor.
 38. The method of claim 5, wherein the glutathioneprecursor is a composition comprising glycine, cystine and a source ofglutamate, together with a source of selenium.
 39. The kit of claim 29,wherein the marker of oxidative stress is a lipid peroxide, pyroglutamicacid or glutathione or a combination thereof.
 40. The kit of claim 29,wherein the marker of oxidative stress is 4-hydroxynonal (4HNE) ormalondialdehyde or a protein modified by 4-HNE or malondialdehyde.
 41. Amethod for determining the amount of antioxidant formulation sufficientto (1) increase glutathione synthesis or re-synthesis in a patient inneed thereof; (2) reduce urine pyroglutamic acid in a patient inneedthereof; (3) reduce urine lipid peroxide in a patient in needthereof; and/or (4) diminish urine lipid peroxide and pyroglutamic acidlevels and concurrently increase blood plasma glutathione levels in apatient in need thereof; the method comprising: (a) collecting a seriesof body fluid samples from the patient, wherein said body fluid samplesare collected prior to the start of treatment, and daily after the startof treatment for about 14 days; (b) measuring the amount of lipidperoxide and/or pyroglutamic acid in said body fluid samples; (c)comparing the amount of lipid peroxide and/or pyroglutamic acid in saidbody fluid samples with that of normal standards; (d) optionallymeasuring the amount of glutathione increase in blood samples; (e)optionally comparing the amount of glutathione in said blood sampleswith that of normal standards; and (f) correlating a change in thelevels of lipid peroxide and/or pyroglutamic acid and optionallyglutathione in the body samples of patients receiving the antioxidantformulation.
 42. The method of claim 41, wherein the levels of lipidperoxide are measured by mass spectrometry, absorption spectrometry,liquid chromatography, thin layer chromatography or a redox reagent. 43.The method of claim 41, wherein the levels of lipid peroxide aremeasured by Wheeler method, iron thiocyanate method, thiobarbituric acidmethod or with a combination of a peroxidase and a hydrogen donor. 44.The method of claim 41, which is a colorimetric assay, a fluorescenceassay, a luminometric assay.
 45. The method of claim 41, wherein theurine levels of lipid peroxide are measured with an immunological assay.46. The method of claim 41, wherein the immunological assay isenzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). 47.The method of claim 41, wherein the urine levels of lipid peroxide aremeasured with one or more antibodies to lipid peroxides.
 48. The methodof claim 41, wherein the measurement of the levels of lipid peroxide isconducted in a fluid phase format, a high throughput format, a teststrip format or a test well format.
 49. The method of claim 41, whereinthe pyroglutamic acid is measured by gas chromatography, massspectrometry, or NMR spectroscopy.
 50. The method of claim 41, whereinthe pyroglutamic acid levels are measured with an antibody topyroglutamic acid.
 51. The method of claim 41, wherein the measurementof the levels of pyroglutamic acid is conducted in a fluid phase format,a high throughput format, a test strip format or a test well format. 52.The method of claim 41, wherein the measurement of levels ofpyroglutamic acid is conducted in a test-strip format or a test-wellformat, wherein the test strip or test well comprises an antibody topyroglutamic acid.
 53. The method of claim 41, wherein the levels oflipid peroxide is measured prior to, concurrently with, or shortly afterthe measurement of urine pyroglutamic acid.
 54. The method of claim 41,further comprising measuring the levels of blood plasma glutathione withan enzymatic or non-enzymatic assay.
 55. The method of claim 41, furthercomprising measuring the levels of blood plasma glutathione with anantibody to glutathione.
 56. The method of claim 41, wherein the bloodplasma and urine samples are collected from a subject or a patient whichis a non-human mammal or a human.
 57. The method of claim 41, comprisingdetecting malondialdehyde (MDA) or 4-hydroxynonenal as a marker of lipidperoxide.
 58. The method of claim 41, wherein the body fluid sample isurine, blood or a combination thereof.
 59. A method for determining anorally anti-oxidative amount of a nutritional or therapeutic compositionsufficient to diminish urine lipid peroxide and pyroglutamic acid levelsand concurrently increase blood plasma glutathione levels, comprisingthe steps of: a) collecting blood plasma and urine samples prior toadministration of the nutritional or therapeutic composition and dailyafter the start of administration for about 14 days; b) measuring theurine levels of lipid peroxide and pyroglutamic acid and blood plasmalevels of glutathione both prior to and after the start ofadministration of the nutritional or therapeutic composition; c)determining whether there is (1) a decrease in lipid peroxide andpyroglutamic acid levels after the administration of the nutritional ortherapeutic composition compared to the levels thereof before theadministration of nutritional or therapeutic composition and (2) anincrease in blood glutathione levels after the administration of thenutritional or therapeutic composition compared to the levels thereofbefore the administration of the nutritional or therapeutic composition;and d) correlating the decrease in lipid peroxide and pyroglutamic acidof (c)(1) with the increase in glutathione levels of (c)(2), whereinsaid correlation establishes an orally anti-oxidative effective amountof the nutritional or therapeutic composition.
 60. The method of claim59, wherein the measurement of the levels of lipid peroxide andpyroglutamic acid is conducted in a fluid phase format, a highthroughput format, a test strip format or a test well format.
 61. Themethod of claim 59, wherein the glutathione precursor is a compositioncomprising glycine, cystine and a source of glutmate, together with asource of selenium.